Method for rapidly evaluating performance of short interfering RNA with novel chemical modifications

ABSTRACT

It is an object of the instant invention to provide a method for the rapid evaluation of novel sugar modifications to be used in siRNA synthesis including the rapid evaluation of chemical modification patterns within the siRNA to effectuate increased stability and ultimately increased efficacy of a siRNA therapeutic. It is a further object of the instant invention to provide novel nucleosides useful for siRNA therapy.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved cellularmechanism of post transcriptional gene silencing found in fungi, plantsand animals that uses small RNA molecules to guide the inhibition ofgene expression in a sequence-specific manner. Its principal role issuppression of potentially harmful genetic material (Buchon et al.,“RNAi: a defensive RNA-silencing against viruses and transposableelements”, Heredity, 96(2), 95-202, 2006). Among the most powerfulstimuli capable of triggering the RNAi machinery are longdouble-stranded ribonucleic acids (dsRNA) often associated with viralreplication. These long duplexes are degraded into short double-strandedfragments (approximately 21-23 nucleotides long) known as smallinterfering RNA (siRNA) by an RNAse III-type enzyme, Dicer (Bernstein etal., “Role for a bidentate ribonuclease in the initiation step of RNAinterference” Nature, 409, 363-366, 2001). The siRNA is then transferredonto the RNA-induced silencing complex (RISC) (Tuschl et al., “RISC is a5′-phosphomonoester-producing RNA endonuclease”, Genes & Development,18: 975-980, 2004) which becomes activated upon removal of one of thestrands (the “passenger” or “sense” strand). The remaining strand (the“guide” or “antisense” strand) then directs the activated RISC in asequence-specific degradation of complementary target messenger RNA(mRNA). Since the selection of engaged mRNA is controlled solely byCrick-Watson base-pairing (Watson, J. D, Crick, F. H “Molecularstructure of nucleic acids”, Nature, (171), 737-738, 1953) between theguide strand and the target mRNA, the RNAi pathway can be directed todestruct any mRNA of a known sequence. In turn, this allows forsuppression, or knock-down, of any gene from which it was generatedpreventing the synthesis of the target protein. This unprecedentedcontrol has wide reaching therapeutic consequences.

While long dsRNA will also inevitably trigger a sequence independentimmunogenic reaction, much smaller siRNA duplexes introduced exogenouslywere found to be equally effective triggers of RNAi (Zamore, P. D.,Tuschl, T., Sharp, P. A., Bartel, D. P.; “RNAi: double-stranded RNAdirects the ATP-dependent cleavage of mRNA at 21 to 23 nucleotideintervals.” Cell, 101, 25-33, 2000). According to this, artificiallysynthesized 21 nucleotide long RNA duplexes typically containing 2-nt3′-overhangs, can be used to manipulate any therapeutically relevantbiochemical system, including ones which are not accessible throughtraditional small molecule control.

In order to realize this immense therapeutic potential of RNAi, manyproperties of siRNAs need to be optimized (Castanotto et al. “Thepromises and pitfalls of RNA interference-based therapeutics”, Nature,457, 426-457, 2009). Due to their large molecular weight and polyanionicnature unmodified siRNAs do not freely cross the cell membranes, andthus development of special delivery systems is required (White, P. J.“Barriers to successful delivery of short interfering RNA after systemicadministration” Clin. Exp. Pharmacol. Physiol. 35, 1371-1376, 2008).Equally important is optimization of potency (Koller, E. et al.“Competition for RISC binding predicts in vitro potency of siRNA” Nucl.Acids Res. 34, 4467-4476, 2006), stability (Damha et al. “Chemicallymodified siRNA: Tools and applications” Drug Discovery Today, 13(19/20),842-855, 2008), and immunogenicity (Sioud “Innate sensing of self andnon-self RNAs by Toll-like receptors”, TRENDS in Molecular Medicine,12(4), 167-176, 2006).

The guide-strand-mediated sequence-specific cleavage activity of theRNA-induced silencing complex (RISC) is associated with an RNase H-typeendonuclease Argonaute (Ago) (Tanaka Hall “Structure and Function ofArgonaute Proteins”, Structure, 13, 1403-1408, 2005). An X-ray crystalstructure of Argonaute 2 (Ago2) containing a chemically modified guidestrand (Patel at al., “Structure of the guide-strand-containingargonaute silencing complex”, Nature, 456(13), 209-213, 2008, Patel atal., “Structure of an argonaute silencing complex with a seed-containingguide DNA and target RNA duplex”, Nature, 456(13), 921-927, 2008)revealed that nucleotides 2 through 8 of the guide strand (referred toas “seed region”) are preassembled in a A-form helix and that the guidestrand makes contact with the surface of the Ago2 through itssugar/phosphodiester backbone. This observation bodes well for theimportance of the seed region in the initial recognition of thecomplementary mRNA, since an effective mRNA/guide strand interactionrequires the heterobases to be accessible from the cytoplasm, hence topoint away from the receptor surface.

Chemical modifications of the sugar/phosphodiester backbone of thesiRNA's guide strand are therefore expected to have profound effect onthe siRNA/Ago2 interaction. This offers a way to optimize theperformance of this complex. Such an improved interaction should resultin increased siRNA/Ago2 binding selectivity, more effective strandselection and passenger strand cleavage, improved catalytic turnover,siRNA/Ago2 complex stability and product release. Moreover, chemicallymodified siRNA duplexes are expected to be quite resistant to RNasemediated cleavage (increased half-life), decreased affinity to Toll-likereceptors (TLR) and dsRNA-dependent protein kinase (PKR), resulting indecreased immunogenicity (Liang et al. “RNA Interference with ChemicallyModified siRNA”, Cur. Topics Med. Chem., 6, 893-900, 2006).

Most chemically modified nucleosides used today in RNAi were synthesizedto convey enzymatic stability to RNA oligomers and their design was notguided by siRNA/Ago2 binding considerations. Even though collection ofSAR data relevant to this interaction would be highly beneficial, ahypothesis driven design of such novel nucleosides is clearly hamperedby the chemical complexity of the associated chemistry. Assuming thatthe interaction of the siRNA's guide strand with the Ago2 surface isprimarily static, such an effort would require independent SAR datacollection for each nucleotide along the siRNA oligomer separately,since the local Ago2 surface relevant to each particular position isdifferent. Furthermore, such SAR study would require the synthesis ofsugar-modified nucleosides containing all four canonical bases since asystematic investigation would require a use of a sequence-specificsiRNA. In theory, at least 21 separate siRNA oligomers, containing oneinstance the modified nucleoside each (positions 1 through 21,“walkthrough”) would be necessary, requiring the synthesis ofconsiderable quantities of each monomer. This complexity renders theinterrogation of such a huge chemical space intractable.

We have realized that use of universal bases in place of canonicalheterocycles would greatly simplify the problem. In general, a universalbase is a heterocycle capable of an isoenergetic interaction with eachone of the canonical heterobases, (Adenine, Guanine, Uracil andCytosine) while part of a double helix. The simplest example of such auniversal interaction is a hydrogen atom corresponding to the removal ofthe heterocycle and more complex examples are 3-nitro-pyrrole,imidazole-4-carboxamide, 5-nitro-indole, inosine and others. We haveargued that the use of such universal base would not only eliminate theneed for synthesis of all four canonical nucleosides, it would alsogreatly reduce the complexity of the associated chemical syntheses.

Replacement of a canonical heterocycle with a universal base wasexpected to affect the efficiency of such a siRNA/Ago2 assembly and thisshould result in a position specific decrease of target gene knockdown.In order to asses this effect, we have synthesized ApoB-specific siRNAoligomers containing such universal bases while keeping the sugar of thenucleoside unmodified. We have indeed observed a base dependent positionspecific change in overall knockdown performance. In general, the effectof the universal base was more pronounced in the center of the RNAoligomer and the effect of simple base-surrogates such as hydrogen(removal of the entire heterocycle) was more profound.

Using the position-specific knockdown data obtained with universalbase-surrogates as the new baseline, we synthesized a series ofsugar-modified base-surrogate containing nucleosides and evaluated themunder conditions similar to those used to obtain the baseline data. Weargued that if the performance of the base-surrogate-containingsugar-modified nucleoside surpasses that of the base-surrogatecontaining canonical sugar, this relationship will be reflected also inthe improved performance of the sugar-modified full canonical nucleosideand vice versa. This approach would allow us to rapidly evaluateprospective sugar modification and perform the syntheses of the fullnucleosides only when the particular modification is found beneficial inthis simplified platform. While this novel platform does not allow for aprediction of useful modifications, it can dramatically increase therate of SAR data collection.

SUMMARY OF THE INVENTION

It is an object of the instant invention to provide a method for therapid evaluation of novel sugar modifications to be used in siRNAsynthesis including the rapid evaluation of chemical modificationpatterns within the siRNA to effectuate increased stability andultimately increased efficacy of a siRNA therapeutic. It is a furtherobject of the instant invention to provide novel nucleosides useful forsiRNA therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C: Position-dependent mRNA degradation of theuniversal bases.

FIG. 2: Efficiency of position-dependent target mRNA degration2′-O-benzyl substituted inosine containing siRNAs.

FIG. 3: Position-dependent relationship between the target mRNAdegradation of siRNA (ApoB 10162) containing the 2-O-benzyl-substitutedinosine and 2′-O-benzyl substituted canonical nucleoside at the sameposition.

FIG. 4: Position-dependent relationship between the target mRNAdegradation of siRNA (ApoB 9514) containing the 2-O-benzyl-substitutedinosine and 2′-O-benzyl substituted canonical nucleoside at the sameposition.

FIG. 5: Evaluation of the 2′-O-benzyl modification on the inosineplatform.

FIG. 6: Evaluation of the 2′-O-ribose modified nucleosides for theirknockdown performance using the inosine platform.

FIG. 7: Evaluation of an ApoB sequence with 2′-O-benzyl modification atpositions 8 and 15; 8, 15, 19; and 5, 8, 15, 19.

FIG. 8: Evaluation of an ApoB sequence with 2′-O-benzyl modification atpositions 5, 8, 13, 15 or 19.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the invention features a method for an efficient andstreamlined process for the evaluation of novel chemical modificationsin an siRNA, said method comprising: a) generating a siRNA oligomer with1 or more sugar un-modified universal base containing nucleosides; b)obtaining position-specific data to create a baseline data; c)generating a siRNA oligomer with 1 or more sugar modified universal basecontaining nucleosides; and d) obtaining position-specific data andcomparing that data to the baseline data.

In an embodiment, the invention features a method for an efficient andstreamlined process for the evaluation of novel chemical modificationsin an siRNA, said method comprising: a) generating a siRNA oligomer with1 or more sugar un-modified universal base containing nucleosides; b)obtaining position-specific knockdown data to create a target geneknockdown baseline data; c) generating a siRNA oligomer with 1 or moresugar modified universal base containing nucleosides; and d) obtainingposition-specific knockdown data and comparing that data to theknockdown baseline data.

In an embodiment, the invention features a method for an efficient andstreamlined process for the evaluation of novel chemical modificationsin an siRNA, said method comprising: a) generating a siRNA oligomer with1 or more sugar un-modified universal base containing nucleosides; b)obtaining position-specific knockdown data to create a target geneknockdown baseline data; c) generating a siRNA oligomer with 1 or moresugar modified universal base containing nucleosides; and d) obtainingposition-specific knockdown data and comparing that data to theknockdown baseline data to determine if the performance of said sugarmodification in that siRNA oligomer is beneficial to knockdownefficiency.

In an embodiment, the invention features a method for an efficient andstreamlined process for the evaluation of novel chemical modificationsin an siRNA, said method comprising: a) generating a siRNA oligomer with1 or more sugar un-modified universal base containing nucleosides,wherein the universal base is a hydrogen atom or inosine; b) obtainingposition-specific data to create a baseline data; c) generating a siRNAoligomer with 1 or more sugar modified universal base containingnucleosides; and d) obtaining position-specific data and comparing thatdata to the baseline data.

In an embodiment, the invention features a method for an efficient andstreamlined process for the evaluation of novel chemical modificationsin an siRNA, said method comprising: a) generating a siRNA oligomer with1 or more sugar un-modified universal base containing nucleosides,wherein the universal base is a hydrogen atom or inosine; b) obtainingposition-specific knockdown data to create a target gene knockdownbaseline data; c) generating a siRNA oligomer with 1 or more sugarmodified universal base containing nucleosides; and d) obtainingposition-specific knockdown data and comparing that data to theknockdown baseline data.

In another embodiment, the invention features a siRNA which contains 1or more 2′-sugar modification(s), wherein the 2′-sugar modification(s)is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains 1or more 2′-sugar modification(s), wherein the 2′-sugar modification(s)is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 3, 5, 6, 8, 15, 17 and 19 whereinthe 2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 3, 5, 6, 8, 15, 17 and 19 whereinthe 2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 3, 5, 6, 8, 15, 17 or 19 whereinthe 2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 3, 5, 6, 8, 15, 17 or 19 whereinthe 2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 5, 8, 15 and 19 wherein the2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 5, 8, 15 and 19 wherein the2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 5, 8, 15 or 19 wherein the2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 5, 8, 15 or 19 wherein the2′-sugar modification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 8, 15 and 19 wherein the 2′-sugarmodification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl; R¹ is independentlyselected from: OH, COOH, CF₃, NO₂, halo, (C₁-C₆)alkyl, O(C₁-C₆)alkyl,aryl, O-aryl, heteroaryl, O-heteroaryl, heterocyclyl or O-heterocyclyl,wherein said alkyl, aryl, heteroaryl and heterocyclyl is optionallysubstituted with from one to three substituents selected from halo and(C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 8, 15 and 19 wherein the 2′-sugarmodification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 8, 15 or 19 wherein the 2′-sugarmodification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a siRNA which contains a2′-sugar modification(s) at positions 8, 15 or 19 wherein the 2′-sugarmodification(s) is represented in Formula B,

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In the embodiment above the siRNA can be a chemically modified siRNA.

In another embodiment, the invention features a compound illustrated bythe Formula A:

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a compound illustrated bythe Formula A:

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl, with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

Specific compounds of the instant invention include:

-   1,4-Anhydro-2-O-benzyl-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)    (dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-tert-butylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-methylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-fluorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-fluorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-fluorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-trifluoromethylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-trifluoromethylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-fluoro-4-trifluoromethylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3,5-difluorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2,6-difluorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2,4-difluorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-difluoromethoxybenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-fluoro-4-chlorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-chlorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-chlorobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-chloro-5-trifluoromethoxybenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-methoxybenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-methoxybenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-methoxybenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-methylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3,5-dimethylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-isopropylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-phenylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-naphthyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(3-nitrobenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(4-(tetrahydropyran-4-yl)benzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-([4-(5-methyl-1,3,4-oxadiazol-2-yl)benzyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-ara-benzyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   1,4-anhydro-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-(2-methylbenzyl)-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol;-   2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(2-methylbenzyl)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(2-naphthyl)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(2-difluoromethoxybenzyl)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(3-methoxybenzyl)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(2-methylbenzyl)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(pyridin-2-ylmethy)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(pyridin-3-ylmethy)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   2′-O-(pyridin-4-ylmethy)-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy(dipropan-2-ylamino)phosphanyl]inosine;-   9-{2-O-(4-bromobenzyl)-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-(benzyl)-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-arabinofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-(4-(pyridine-4-yl)benzyl)-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(3-methyl-1,2,4-oxadiazol-5-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(1,3-oxazol-2-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(1,3-thiazol-5-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(furan-2-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(2-methoxypyridin-3-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(2-methyl-pyrazol-4-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(2-methylpyrrol-2-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(2-(pyrrolidin-1-yl)pyrimidin-5-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[4-(2-methoxypyridin-5-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-(2-bromobenzyl)-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   9-{2-O-[2-(pyridin-4-yl)benzyl]-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-b-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine;-   N-acetyl-2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)    (dipropan-2-ylamino)phosphanyl]adenosine;-   2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]uridine;-   2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-N-(2-methylpropanoyl)-guanosine;    and-   N-Acetyl-2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-N-(2-methylpropanoyl)-cytidine;    or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a compound illustrated bythe Formula B:

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a compound illustrated bythe Formula B:

wherein,

p is 0, 1, 2 or 3;

Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl with the provisothat when aryl is phenyl, then p is not 0;

R¹ is independently selected from: OH, COOH, CF₃, NO₂, halo,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl,heterocyclyl or O-heterocyclyl, wherein said alkyl, aryl, heteroaryl andheterocyclyl is optionally substituted with from one to threesubstituents selected from halo and (C₁-C₆)alkyl;

R² is selected from: a canonical or universal base; and

R³ is selected from H and (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt or stereoisomer thereof.

Specific compounds of the instant invention include:

-   1,4-Anhydro-2-O-benzyl-D-ribitol;-   1,4-Anhydro-2-O-(4-tert-butylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(4-methylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-fluorobenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(4-fluorobenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(3-trifluoromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(4-trifluoromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-fluoro-4-trifluoromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(3,5-ditrifluoromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2,6-difluoromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2,4-difluoromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-difluoromethoxybenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-fluoro-4-chloromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-chloromethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(3-chloro-5-trifluoromethoxybenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(4-methoxybenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(3-methoxybenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-methoxybenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-methylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(3,5-dimethylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(4-isopropylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(4-phenylbenzyl)-D-ribitol;-   1,4-Anhydro-2-O-(2-naphthyl)-D-ribitol;-   1,4-Anhydro-2-O-(3-nitrobenzyl)-D-ribitol;-   1,4-Anhydro-2-O-[(4-tetrahydropyran-4-yl)benzyl]-D-ribitol;-   1,4-Anhydro-2-O-[(5-methyl-1,2,4-oxadiazol-3-yl)benzyl]-D-ribitol;-   1,4-Anhydro-2-O-[(5-methyl-1,3,4-oxadiazol-2-yl)benzyl]-D-ribitol;-   1,4-Anhydro-2-O-benzyl]-D-arabinitol;-   1,4-Anhydro-2-O-(2-methylbenzyl)-D-ribitol;-   2′-O-benzylinosine;-   2′-O-(2-methylbenzyl) inosine;-   2′-O-(2-naphthyl) inosine;-   2′-O-(2-difluoromethoxybenzyl) inosine;-   2′-O-(3-methoxybenzyl) inosine;-   2′-O-(pyridin-2-ylmethyl) inosine;-   2′-O-(pyridin-3-ylmethyl) inosine;-   2′-O-(pyridin-4-ylmethyl) inosine;-   2′-O-(4-bromobenzyl) inosine;-   9-(2-O-benzyl-b-D-arabinofuranosyl)-1,9-dihydro-6H-purin-6-one-   2′-O-[(4-pyridin-4-yl)benzyl]inosine;-   2′-O-[4-(5-methyl-1,3,4-oxadiazol-2-yl)benzyl]inosine;-   2′-O-[4-(oxazol-2-yl)benzyl]inosine;-   2′-O-[4-(thiazol-2-yl)benzyl]inosine;-   2′-O-[4-(furan-2-yl)benzyl]inosine;-   2′-O-[4-(2-methoxypyridin-3-yl)benzyl]inosine;-   2′-O-[4-(2-methylpyrazol-4-yl)benzyl]inosine;-   2′-O-[4-(1-methylpyrol-2-yl)benzyl]inosine;-   2′-O-[4-(5-methyl-1,2,4-oxadiazol-3-yl)benzyl]inosine;-   2′-O-{4-[2-(pyrrolidin-1-yl)pyrimidin-5-yl]benzyl}inosine;-   2′-O-[4-(2-methoxypyridin-5-yl)benzyl)]inosine;-   2′-O-[4-(3-fluoropyridin-5-yl)benzyl]inosine;-   2′-O-(2-bromobenzyl) inosine;-   2′-O-(pyridyn-4-ylmethyl) inosine;-   2′-O-[2-(2-methoxypyridin-5-yl)benzyl]inosine;-   N-acetyl-2′-O-benzyladenosine;-   2′-O-benzyluridine;-   2′-O-benzylguanosine; and-   N-Acetyl-2′-O-benzylcytidine;    or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a siRNA which contains 1or more 2′-sugar modification(s), wherein the 2′-sugar modification(s)are represented in Tables 1-3.

DEFINITIONS

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, all such stereoisomers beingincluded in the present invention.

It is understood that one or more silicon (Si) atoms can be incorporatedinto the compounds of the instant invention in place of one or morecarbon atoms by one of ordinary skill in the art to provide compoundsthat are chemically stable and that can be readily synthesized bytechniques known in the art from readily available starting materials.Carbon and silicon differ in their covalent radius leading todifferences in bond distance and the steric arrangement when comparinganalogous C-element and Si-element bonds.

Regarding the compounds of the instant invention, it is understood thatthe atoms may exhibit their natural isotopic abundances, or one or moreof the atoms may be artificially enriched in a particular isotope havingthe same atomic number, but an atomic mass or mass number different fromthe atomic mass or mass number predominantly found in nature. Thepresent invention is meant to include all suitable isotopic variationsof the compounds. For example, different isotopic forms of hydrogen (H)include protium (¹H) and deuterium (²H). Protium is the predominanthydrogen isotope found in nature. Enriching for deuterium may affordcertain therapeutic advantages, such as increasing in vivo half-life orreducing dosage requirements, or may provide a compound useful as astandard for characterization of biological samples.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁-C₆, as in“(C₁-C₆)alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6carbons in a linear or branched arrange-ment. For example,“(C₁-C₆)alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, i-butyl, pentyl, hexyl, and so on.

The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbongroup having the specified number of carbon atoms. For example,“cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydro-naphthyl, indanyl and biphenyl. In cases where thearyl substituent is bicyclic and one ring is non-aromatic, it isunderstood that attachment is via the aromatic ring.

The term “heteroaryl”, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition ofheterocycle below, “heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Suchheteraoaryl moieties for substituent Q include but are not limited to:2-benzimidazolyl, 2-quinolinyl, 3-quinolinyl, 4-quinolinyl,1-isoquinolinyl, 3-isoquinolinyl and 4-isoquinolinyl.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 3- to 10-membered aromatic or nonaromatic ring containing from 1to 4 heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes the abovementioned heteroaryls, as well as dihydro and tetrathydro analogsthereof. Further examples of “heterocyclyl” include, but are not limitedto the following: benzoimidazolyl, benzoimidazolonyl, benzofuranyl,benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl,indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl,pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl,dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro (Cl), fluoro (F), bromo (Br) andiodo (I).

“siRNA oligomer” means a nucleic acid molecule capable of mediating RNAi(RNA interference). siRNA oligomers (or siRNAs) are well known in theart.

“Sugar un-modified universal base containing nucleoside(s)” means anucleoside, wherein the canonical base (Adenine, Guanine, Cytosine orUracil) is replaced with a universal base.

“Sugar modified universal base containing nucleoside(s)” means anucleotide, wherein the canonical base (Adenine, Guanine, Cytosine orUracil) is replaced with a universal base and the ribose is chemicallymodified.

“Universal base” means a group (typically a heterocycle) which iscapable of isoenergetic interaction with a naturally occurring base(Loakes “Survey and summary: The application of universal DNA basedanalogues”, Nuc. Acids Res., 29(12), 2437-2447, 2001). Examples ofuniversal bases include but are not limited to a hydrogen atom, inosine,modified adenine, modified guanine, modified uracil, modified cytosine,thymine, modified thymine, 2-aminoadenosine, 5-methylcytosine,2,6-diaminopurine, or any base that can be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, imidazole-4-carboxamide,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

“Canonical base” means adenine, guanine, uracil or cytosine.

“Position-specific data” means any data generated to establish siRNAoligomer function, wherein the siRNA oligomer contains 1 or more sugarmodified or unmodified universal or canonical base containingnucleoside(s) at a specific position of the siRNA oligomer.

“Baseline data” means any data generated to establish siRNA oligomerfunction, wherein the siRNA oligomer contains 1 or more sugarun-modified universal base containing nucleoside(s).

“Position-specific knockdown data” means any knockdown data generated toestablish siRNA oligomer function, wherein the siRNA oligomer contains 1or more sugar modified or un-modified universal or canonical basecontaining nucleoside(s) at a specific position of the siRNA oligomer.

“Knockdown baseline data” means any knockdown data generated toestablish siRNA oligomer function, wherein the siRNA oligomer contains 1or more sugar un-modified or un-modified universal or canonical basecontaining nucleoside(s).

“Chemically modified siRNA” means an siRNA with chemical modificationsthat include phosphorothioate linkages, 2′-hydroxyl groups (including2′-O-methyl, 2′-fluoro, 2′-O-(2-methoxyethyl and locked nucleic acid),base modifications, terminal nucleotide(s) modification and others.

In an embodiment, p is 0, 1 or 2.

In an embodiment, p is 0 or 1.

In an embodiment, p is 0.

In an embodiment, Ring K is aryl, heterocyclyl or a (C₃-C₈)cycloalkyl,with the proviso that Ring K is not phenyl.

In an embodiment, Ring K is phenyl, naphthyl or pyridyl.

In an embodiment Ring K is phenyl.

In an embodiment R¹ is OH, COOH, CF₃, NO₂, halo, (C₁-C₆)alkyl,O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl, heterocyclyl orO-heterocyclyl, wherein said alkyl, aryl, heteroaryl and heterocyclyl isoptionally substituted with from one to three substituents selected fromO(C₁-C₆)alkyl, heterocyclyl, halo and (C₁-C₆)alkyl.

In an embodiment R¹ is OH, COOH, CF₃, NO₂, halo, (C₁-C₆)alkyl,O(C₁-C₆)alkyl, aryl, O-aryl, heteroaryl, O-heteroaryl, heterocyclyl orO-heterocyclyl, wherein said alkyl, aryl, heteroaryl and heterocyclyl isoptionally substituted with from one to three substituents selected fromhalo and (C₁-C₆)alkyl.

In an embodiment R¹ is OH, COOH, CF₃, NO₂, halo, (C₁-C₄)alkyl,O(C₁-C₆)alkyl, phenyl, O-phenyl, O-pyranyl or oxadiazolyl, wherein saidalkyl, phenyl, and oxadiazolyl is optionally substituted with from oneto three substituents selected from halo and (C₁-C₄)alkyl.

In an embodiment R² is adenine, guanine, uracil or cytosine.

In an embodiment R² is a universal base.

In an embodiment R² is a hydrogen atom or inosine.

In an embodiment R³ is selected from H and CH₃.

In an embodiment R³ is H.

In an embodiment, the universal base is a hydrogen atom, or inosine.

In an embodiment, the ribose is chemically modified at the 2′-position.

Examples Method

General Synthesis of Sugar-Modified Nucleoside Phosphoramidites.

The synthetic relay to access the modified anhydroribitol amidites,Intermediate 1, was prepared following the procedure depicted in Scheme1.

Commercial grade ribose (1-1) was converted to the respective2′,3′-O-acetonide 1-2 and the less hindered primary hydroxyl in 1-2 wasprotected as a tert-butyl-diphenylsilyl ether (1-3) following apreviously described procedure (Choi, W. J. et al.: “Preparative andStereoselective Synthesis of the Versatile Intermediate for CarbocyclicNucleosides: “Effects of the Bulky Protecting Groups to Enforce FacialSelectivity”, J. Org. Chem., 69, 2634-2636, 2004). Acetal 1-3 exists inequilibrium with the respective ring-open aldehyde and its exposure tosodium borohydride led to ring open diol 1-4. Monosilylation of thisdiol at the less hindered primary hydroxyl was achieved under standardconditions, and the ring-closure was affected at elevated temperature inthe presence of a suitable base. The silyl-based protecting grouppresent in 4-6 was removed using standard exposure to stoichiometricquantities of TBAF at ambient temperature, or at elevated temperatureusing excess potassium fluoride and TBAF catalysis. The penultimateintermediate 1-8 was obtained by careful treatment of alcohol 1-7 with85% trifluoroacetic acid at 0 C and the synthesis was completed byselective protection of the 1,3-diol in 1-8 using thedi-tert-butylsilylene protecting group (Greene, T. W., Wuts, P. G. M.,“Protection of 1,2 and 1,3 diols”, p. 237 in “Protective groups inorganic synthesis”, A Wiley Interscinece Publication, third edition,1999).

Intermediate 2 was synthesized in a five step synthetic sequence asdescribed in Scheme 2. According to this, inosine (2-1) was protected,as usual for 1,3-diols, with di-tert-butylsilylene protecting group(Araki, L. et al., “Synthesis of novel C4-linked C2-ImidazoleRibonucleotide and its Application in Probing the catalytic Mechanism ofa Ribozyme”, J. Org. Chem., 74, 2350-2356, 2009). To control theregioselectivity of the alkylation, the C6-carbonyl group was protectedas a trimethylsilylethyl ether. This was achieved as follows: Thesecondary hydroxyl in 2-2 was temporarily protected as an acetyl ester(2-3) and the amide group within hypoxanthine was activated with2,4,6-triisopropyl phenylsulfonyl chloride (compound 2-4). Reaction of2-4 with trimethylsilyl ethanol in a presence of a base led to formationof 2-5. The acetate temporary protecting group present in 2-5 wasremoved using standard basic conditions.

The 2′-O-benzyl modified anhydroribitol phosphoramidites weresynthesized utilizing a four step synthetic sequence starting fromIntermediate 1 as shown in Scheme 3.

According to this, Intermediate 1 was alkylated with the appropriatebenzyl bromide in the presence of a base to produce the derivative 3-1.The silyl protecting group was cleaved using tetrabutylammonium fluorideand the obtained polar diol 3-2 was purified using preparative HPLCutilizing a mass-spectrometric detector. The last two steps could alsobe performed without isolation of intermediate 3-1. The primary hydroxylin diol 3-2 was then protected as a dimethoxytrityl ether by reacting3-2 with dimethoxytrityl chloride at ambient temperature in a presenceof a suitable base. The final phosphoamidites were then obtained by areaction of the alcohol 3-3 with diisopropylamino-cyanoethoxyphosphorouschloride at ambient temperature and in the presence of a base.

A similar procedure was used to synthesize the appropriate 2′-O-modifiedinosine amidites, and the sequence is summarized in Scheme 4.

According to this, Intermediate 2 was alkylated with the appropriatebenzyl bromide in a presence of a suitable base and the intermediate 4-1was exposed to tetrabutylammonium fluoride at ambient temperature toaffect simultaneous cleavage of both silyl-containing protecting groups.Alternatively, catalytic quantity of TBAF could be utilized if excess ofKF was applied at elevated temperature. The diol 4-2 was then purifiedunder usual conditions of column chromatography and the primary alcoholwas protected as a dimethoxytrityl ether (4-3) as described above. Thefinal amidites were then obtained by reacting intermediate 4-3 withdiisopropylamino cyanoethoxyphosphorous chloride at ambient temperatureand in the presence of an appropriate base. Final purifications wereaffected by column chromatography on triethylamine pretreated silicagel.

2′-O-benzyl substituted inosine amidites carrying a aromatic ring at theortho- or para-positions could be readily accessed from downstreamsynthetic relays such as 5-3 through cross-coupling reactions, Scheme 5.According to this, intermediate 2 was alkylated with ortho- orpara-bromobenzyl bromide as described above (Scheme 4), the cyclic silylprotecting group in 5-1 was then selectively removed using HF.Pyridine.The primary hydroxyl in 5-2 was protected as 4,4′-dimethoxytritylether(5-3) and these relays (general structure 5-3, para-bromobenzylderivative, Intermediate 3 or, ortho-bromobenzyl derivative Intermediate4), were further functionalized using aryl boron (Miyaura, Norio;Yamada, Kinji; Suzuki, Akira (1979) “A new stereospecific cross-couplingby the palladium-catalyzed reaction of 1-alkenylboranes with 1-alkenylor 1-alkynyl halides”. Tetrahedron Letters 20 (36): 3437-3440) or tin(Milstein, D.; Stille, J. K. J. Am. Chem. Soc. 1978, 100, 3636) basedcross-coupling chemistry. The final amidites 5-5 were then synthesizedas described above.

The 2′-O-substituted anhydroribitols, synthesis of which is depicted inScheme 3, are summarized in Table 1, the 2′-O-substituted inosines,described in Scheme 4 and 5 are summarized in Table 2 and 3.

TABLE 1

Found Entry Structure (R) Empirical formula Calculated [M + H]⁺  1

C₄₂H₅₁N₂O₇P 726.3 727.4  2

C₄₆H₅₉N₂O₇P 782.4 783.2  3

C₄₃H₅₃N₂O₇P 740.4 741.5  4

C₄₂H₅₀FN₂O₇P 744.3 745.3  5

C₄₂H₅₀FN₂O₇P 740.4 745.3  6

C₄₂H₅₀FN₂O₇P 740.4 745.3  7

C₄₃H₅₀F₃N₂O₇ 794.3 795.2  8

C₄₃H₅₀F₃N₂O₇ 794.3 795.3  9

C₄₃H₄₉F₄N₂O₇ 812.3 813.3 10

C₄₂H₄₉F₂N₂O₇ 762.3 763.3 11

C₄₂H₄₉F₂N₂O₇ 762.3 763.3 12

C₄₂H₄₉F₂N₂O₇ 762.3 763.3 13

C₄₃H₅₁F₂N₂O₈ 792.3 793.4 14

C₄₂H₄₉ClFN₂O₇P 778.3 779.3 15

C₄₂H₅₀ClN₂O₇P 760.3 761.4 16

C₄₂H₅₀ClN₂O₇P 760.3 761.2 17

C₄₃H₄₉ClF₃N₂O₈P 844.3 845.2 18

C₄₃H₅₃N₂O₈P 756.4 757.4 19

C₄₃H₅₃N₂O₈P 756.4 757.4 20

C₄₃H₅₃N₂O₈P 756.4 757.3 21

C₄₃H₅₃N₂O₇P 740.4 741.4 22

C₄₄H₅₅N₂O₇P 754.4 755.4 23

C₄₅H₅₇N₂O₇P 768.4 769.4 24

C₄₈H₅₅N₂O₇P 802.4 803.4 25

C₄₆H₅₃N₂O₇P 776.4 777.3 26

C₄₂H₅₀N₃O₉P 771.3 772.2 27

C₄₇H₅₉N₂O₈P 826.4 827.3 28

C₄₅H₅₃N₄O₈P 808.4 809.4 29

C₄₅H₅₃N₄O₈P 808.4 809.5 30

C₄₂H₅₁N₂O₇P 726.3 727.2 31

C₄₃H₅₃N₂O₇P 740.4 741.4

TABLE 2

Empirical Calcu- Entry Structure (R) formula lated Found 1

C₄₇H₅₃N₆O₈P 860.43 861.3 2

C₄₈H₅₅N₆O₈P 874.4  875.5 3

C₅₁H₅₅N₆O₈P 910.4  911.5 4

C₄₈H₅₃F₂N₆O₉P 926.4  927.4 5

C₄₈H₅₅ClN₆O₉P 890.4  891.5 6

C₄₆H₅₂N₇O₈P 861.4  862.4 7

C₄₆H₅₂N₇O₈P 861.4  862.4 8

C₄₆H₅₂N₇O₈P 861.4  862.4

TABLE 3

Entry Structure (R) Empirical formula Calculated Found  1

C₅₂H₆₄BrN₆O₈PSi 1038.3 1039.4  2

C₅₂H₆₅N₆O₈PSi  961.1  961.6  3

C₅₇H₆₈N₄O₈PSi 1037.5 1038.2  4

C₅₅H₆₇N₈O₉PSi 1042.5 1043.5  5

C₅₅H₆₆N₇O₉PSi 1027.4 1028.5  6

C₅₅H₆₆N₇O₈PSSi 1043.4 1044.4  7

C₅₆H₆₇N₆O₉PSi 1026.4 1027.5  8

C₅₈H₇₀N₇O₉PSi 1067.5 1068.5  9

C₅₆H₆₉N₈O₈PSi 1040.5 1041.5 10

C₅₇H₇₀N₇O₈PSi 1039.5 1040.5 11

C₅₅H₆₇N₈O₉PSi 1042.5 1043.5 12

C₆₀H₇₄N₉O₈PSi 1107.5 1108.5 13

C₅₈H₇₀N₇O₉PSi 1067.5 1068.5 14

C₅₇H₆₇FN₇O₈PSi 1055.5 1056.5 15

C₅₂H₆₄BrN₆O₈PSi 1038.3 1039.5 16

C₅₇H₆₈N₄O₈PSi 1037.5 1038.5Solid-Phase Synthesis of Oligonucleotides.

Synthesis of oligonucleotides from natural or chemically modifiednucleoside phosphoramidites (phosphoramidites) proceeds in a synthesiscycle where a series of 4 chemical steps is repeated, varying the natureof the phosphoramidite, to obtain the requisite oligonucleotidesequence. The synthesis is carried out on an automated synthesizer fromthe 3′- to 5′-end on a solid support, typically controlled pore glass(CPG) or polymer bead, where the first 3′ protected nucleoside isattached to the support via a suitable linker, often comprising asuccinyl linkage.

The first step in the synthesis cycle is removal of a 5′-hydroxyldimethoxytrityl (DMT) protecting group by treatment with a suitableacid, such as trichloroacetic acid or dichloroacetic acid. Followingdeprotection, the second step is chain elongation by coupling with asuitably protected phosphoramidite in the presence of an activator, suchas 5-Ethylthio-1H-Tetrazole. The third step in the synthesis cycle isoxidation of phosphorous using an oxidizer, such as iodine in pyridine.Finally, the fourth step is capping any remaining uncoupled 5′-hydroxylgroups with a suitable agent, such as acetic anhydride. This synthesiscycle is repeated; varying the nature of the phosphoramidite, until thedesired chain length and composition has been prepared.

The protected oligonucleotide is then liberated from the solid phasesupport by treatment with a suitable base, such as methylamine in water,which can affect both the cleavage of the oligonucleotide chain from thesolid support, as well as remove any exocyclic amine protecting groups.If the oligonucleotide contains any 2′-O-silyl protecting groups, thesemay be removed by treatment with a suitable reagent, such astriethylamine trihydroflouride.

The crude 5′-DMT protected oligonucleotide is then purified on asuitable support, such as a C-18 resin, where deprotection by-productsare removed by elution with aqueous solvents, short-chain cappedoligonucleotides may be removed with buffer elution, and the final5′-DMT protecting group is removed by treatment with an acid, such astrifluoroacetic acid. The purified oligonucleotide is then eluted usinga suitable mixture of an organic solvent in water.

This process is repeated to prepare a suitably complimentaryoligonucleotide strand.

The two complimentary strands are then mixed together in a 1:1 molarratio to afford an siRNA duplex.

Biological Evaluation of siRNA Oligonucleotides and Results.

RNA oligomers were evaluated for their siRNA performance using the fivesequences shown in Table 3.

TABLE 3 Position in Gene mRNA sequence Guide strand sequence (5′-3′)ApoB 9514 AUUUCAGGAAUUGUUAAAG (SEQ. ID. NO.: 1) ApoB 10162UUCAGUGUGAUGACACUUG (SEQ. ID. NO.: 2) PCSK9 1965 AAUGCAUUGAGGGCCUUGC(SEQ. ID. NO.: 3) PHD2 196 AUCAAAUUUGGGUUCAAUG (SEQ. ID. NO.: 4) PHD2384 UAGACGUCUUUGCUGACUG (SEQ. ID. NO.: 5)

Positions 1-19 of both strands were ribonucleotides, and the overhangsat positions 20 and 21 contained 2′-deoxyribonucleotide thymidines. Thisunmodified siRNA was the template for systematic evaluation of modifiedsiRNAs containing a single modification at every position along theguide strand.

First, we systematically examined the position-dependent effect of thesugar-unmodified universal base on gene knockdown. This was accomplishedusing the following “modification walkthrough”. In order to examine thiseffect for the ApoB 9514 sequence, i.e. AUUUCAGGAAUUGUUAAAG (SEQ.ID.NO.:1), we synthesized this RNA oligomer with its first nucleotide,adenosine (A), replaced with anhydroribitol, or inosine (X). Then, asecond sequence, in which the second nucleoside (U) was replaced withanhydroribitol or inosine was synthesized, keeping all other nucleotidesunchanged. Altogether nineteen sequences were synthesized where theuniversal base replaced all the natural nucleosides in that sequence.This “modification walkthrough” is depicted in Table 4.

TABLE 4 Position in Guide strand Entry Gene mRNA sequencesequence (5′-3′) unmodified ApoB 9514 AUUUCAGGAAUUGUUAAAG 1 ApoB 9514XUUUCAGGAAUUGUUAAAG 2 ApoB 9514 AXUUCAGGAAUUGUUAAAG 3 ApoB 9514AUXUCAGGAAUUGUUAAAG 4 ApoB 9514 AUUXCAGGAAUUGUUAAAG 5 ApoB 9514AUUUXAGGAAUUGUUAAAG 6 ApoB 9514 AUUUCXGGAAUUGUUAAAG 7 ApoB 9514AUUUCAXGAAUUGUUAAAG 8 ApoB 9514 AUUUCAGXAAUUGUUAAAG 9 ApoB 9514AUUUCAGGXAUUGUUAAAG 10 ApoB 9514 AUUUCAGGAXUUGUUAAAG 11 ApoB 9514AUUUCAGGAAXUGUUAAAG 12 ApoB 9514 AUUUCAGGAAUXGUUAAAG 13 ApoB 9514AUUUCAGGAAUUXUUAAAG 14 ApoB 9514 AUUUCAGGAAUUGXUAAAG 15 ApoB 9514AUUUCAGGAAUUGUXAAAG 16 ApoB 9514 AUUUCAGGAAUUGUUXAAG 17 ApoB 9514AUUUCAGGAAUUGUUAXAG 18 ApoB 9514 AUUUCAGGAAUUGUUAAXG 19 ApoB 9514AUUUCAGGAAUUGUUAAAX (X represents a universal base, such asanhydroribitol, inosine or 5-nitroindole incorporated intoAUUUCAGGAAUUGUUAAAG (SEQ. ID. NO.: 1))

Similar evaluation of universal bases was performed using all fivesequences shown in Table 3 and their mRNA degradation was established asfollows: In a 96-well format, Hepa1-6 cells were transfected with eitherthe unmodified, modified, or negative control siRNA using a commerciallipid transfection reagent. The target mRNA was assessed for degradationusing standard Taqman procedures. The position-dependent mRNAdegradation of the universal bases will be demonstrated using ApoB 9514,with the results summarized in FIGS. 1A, 1B and 1C.

In general, replacement of natural nucleosides with inosines oranhydroribitols resulted in target mRNA degradation. The two universalnucleosides which are not forming Watson-Crick base pairs showed reducedtarget mRNA degradation in the central region (positions 9 to 13). Theactivity was satisfactory when nucleosides at positions 14 through 19were replaced with ones containing universal bases. Overall, the bestperforming universal nucleoside was found to be inosine, FIG. 1C.

The mRNA degradation levels (e.g. those in FIG. 1C) observed with siRNAswhere X is a universal base were used as baselines to assess modifiedinosines or modified anhydroribitols at the same positions. Theusefulness of a particular nucleoside modification was evaluated byincorporation of a modified universal base containing nucleoside (X)into a RNA sequence such as depicted in Table 4. If the target mRNAdegradation of the siRNA containing the modified universal nucleosidewas better than that observed for the same siRNA containing theunmodified universal nucleoside at the same position, it was concludedthat this modification is beneficial at that position. This isexemplified using 2′-O-benzyl modified inosines in FIG. 2.

The efficiency of a position-dependent target mRNA degration 2′-O-benzylsubstituted inosine containing siRNAs (FIG. 2) suggests that a single2′-O-benzyl substitution is well tolerated throughout the seed region,particularly at positions 4, 6, 7, 8 and 9. The target mRNA degradationdata recorded for the 2′-O-benzyl-modified canonical bases confirm thatthis modification is well tolerated throughout the seed region,particularly at positions 6, 8 and 9, see FIG. 2.

The position-dependent relationship between the target mRNA degration ofsiRNA (ApoB 10162) containing the 2-O-benzyl-substituted inosine and2′-O-benzyl substituted canonical nucleoside at the same position isdemonstrated using a logarithmic representation of mRNA degradation(ddCt, Y-axis) FIG. 3. The modified inosine is normalized to the inosineat the same position. The 2′-O-benzyl modified nucleoside can be placedin an siRNA oligomer at positions 5, 6, 8, 14 and 19 and is welltolerated. A single 2′-O-benzyl substituted uridine at position 8 of theApoB 10162 will result in slight improvement of the knockdownefficiency. Placing a 2′-O-benzyl-uridine at position 2, or a2′-O-benzyl-cytidine at position 14 of this siRNA will have negativeimpact on knockdown efficiency.

Similar conclusions can be drawn comparing the behaviour of 2′-O-benzylinosine to that of 2′-O-benzyl substituted canonical nucleoside in adifferent sequence targeting the same gene (ApoB 9514), FIG. 4.Placement of a 2′-O-benzyl substituted canonical nucleoside at position5, 8 and 15 is well tolerated, while the same modification at position 2and 14 is detrimental to performance.

Evaluating the 2′-O-benzyl modification on the inosine platform usingthe PCSK9 (1965) sequence suggests, that this particular modificationwill be well tolerated at positions 3, 6, 8, 13, 15 and 19, see FIG. 5.Indeed, placing 2′-O-benzyl modified nucleosides at positions 3, 6, 8,13, 15 and 19 led to retention of silencing activity when compared tothe unmodified sequence. In fact, as seen previously, 2′-O-benzylsubstituents at positions 8 and 15 lead to slight improvements ofactivity.

The above data indicates that knockdown performance of a particularlymodified inosine containing RNA oligomer can be used to assess thepotential behaviour of the similarly modified full nucleoside. A numberof 2′-O-ribose modified nucleosides were evaluated for their knockdownperformance in the ApoB 10162 sequence using the inosine platform andthe collected data are shown in FIG. 6. The included the2′-O-methylene-(β-naphthyl), Entry 4, Table2,2′-O-(2-difluoromethoxybenzyl), Entry 5, Table 2 and2′-O-methylene-(4-pyridyl), Entry 9, Table 2.

Once again, the collected data suggest that the benzyl modifiednucleosides can be tolerated within the seed region at positions 5 and8, as well as position 15. In addition, the basic pyridine derivative(Entry 9, Table 2) appears to be tolerated at position 3.

Placement of a 2′-O-benzyl substituted canonical nucleoside at position5, 8, 15, or 19 is well tolerated. Combinations of these positions wereevaluated for mRNA degration activity. FIG. 7 shows an ApoB sequencewith 2′-O-benzyl in combination at positions 8 and 15; 8, 15, 19; and 5,8, 15, 19. There is a 4-fold increased in activity over the samesequence containing ribose at those same positions at 1 nM, indicatingcombinations of these modifications lead to improvement in activity.

ApoB siRNAs corresponding to unmodified or modified with 2′-O-benzyl atpositions 5, 8, 13, 15 or 19 were formulated into lipid nanoparticlesand delivered by intravenous (i.v.) tail injection into mice (FIG. 8).2′-O-Benzyl at positions 5, 13, 15, or 19 had similar activity tounmodified in vivo. Position 8 containing a 2′-O-Benzyl had astatistically significant increase in its ability to induce mRNAdegradation from unmodified. The collected data suggest that the benzylmodified nucleosides can be tolerated within an siRNA and improve mRNAdegradation in vivo.

Assays

Transfection and qPCR: Hepa1-6 cells were cultured in Dubellco'sModified Eagle Medium (Mediatech Cellgro) containing 10% serum. Cellswere plated in a 96-well plate (3,500 cells/well) and were transfectedtwenty-four hours after plating in Opti-MEM I Reduced Serum Media(Gibco) and Lipofectamine RNAiMax reagent (Invitrogen) for a finalconcentration of 10 nM siRNA for the initial screening. For IC50 curvesthe final concentration ranged from 0.15 pM to 160 nM along a 12 pointtitration curve. Approximately twenty-four hours after transfection,cells were washed with phosphate buffered saline and lysed inCells-to-CT Lysis Buffer (Ambion) with rDNase I (RNase-Free) added. StopSolution (Ambion) was used to halt the reaction. RT-PCR was performedusing 7 uL of cell lysate in 2× RT Buffer with 20× RT Enzyme Mix(Ambion) added. Conditions were as follows: 37° C. for 60 minutes and95° C. for 5 minutes. ApoB mRNA levels were detected by quantitative PCRwith ApoB specific probes from Applied Biosystems. All cDNA samples wereadded to a 10 uL reaction volume with the following cycling conditions:2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. qPCR was assayed using an ABIPrism 7900 sequence detector using 2× Taqman Gene Expression Master Mix(Applied Biosystems). GAPDH mRNA levels were used for datanormalization.

C57BL/6 male mice 20-23 g were purchased from Taconic Farms, Inc. Afterreceiving, the mice rested for a week; were divided in groups of fourand injected intravenously with 200 μl containing 3 mg/kg siRNAformulated in lipid nanoparticles (Pei, Y et al, RNA, 16, 2010). Micewere sacrificed at 72 hours following siRNA injection. Livers wereharvested and processed to assess ApoB mRNA levels by qPCR as describedabove.

Examples Compounds

All non-hydrolytic reactions, unless indicated otherwise were carriedout in dry solvents purchased from Aldrich. HPLC analyses, except forthe amidites, were performed at 60 C using an Agilent Zorbax EclipsePlus C18, 2.1×50 mm, 1.8 micron column, at 0.8 mL/min flow rate, elutedwith a gradient (5 to 95%) of acetonitrile and water with formic acid(0.1%) as a modifier. The amidites were analyzed using a SupelcoAscentis C18, 100×4.6 mm, 2.7 micron column and ammonium formate (3 mM)as a modifier, under otherwise identical conditions. UV traces wererecorded at 220 nm and mass spectra were obtained using an AgilentTechnologies 6140 Quadrupole LC/MS mass spectrometer in both positiveand negative ion mode. NMR spectra were recorded on a Varian Unity 600,500, or 400 spectrometers.

Intermediate 1

Step A 5-O-[tert-Butyl(diphenyl)silyl)-2,3-O-(1-methylethylidene)-D-glycero-pentofuranose

A solution of 2,3-O-(1-methylethylidene)-D-glycero-pentofuranose (40 g,210 mmol), preparation of which is described in Choi, W. J. et al.:“Preparative and Stereoselective Synthesis of the Versatile Intermediatefor Carbocyclic Nucleosides: “Effects of the Bulky Protecting Groups toEnforce Facial Selectivity”, J. Org. Chem., 69, 2634-2636, 2004, anddimethylaminopyridine (0.20 g, 1.637 mmol) in pyridine (300 mL) wastreated dropwise with tert-butyldiphenylsilyl-chloride (54 mL, 57.8 g,210 mmol) and the resulting solution was stirred at ambient temperatureovernight. The solvent was removed in vacuo, and the residue wasdistributed between water (500 mL) and DCM (500 mL). The organic layerwas washed with water, dried with anhydrous magnesium sulfate, filteredand evaporated to dryness. The oily residue (91.5 g) was purified bygradient flash column chromatography using a mixture of hexanes andethyl acetate to obtain 58.9 g (137 mmol, 65%) of the pure product.LCMS: for C₂₄H₃₁O₄Si calculated 411.2. found 411.2 [M+H-H₂O]⁺ and 427.2[M−H]⁻. ¹H NMR (600 MHz, CD₃CN) δ: 7.70 (m, 4H), 7.45 (m, 6H), 5.20 (d,J=5.9 Hz, 1H), 4.75 (d, J=5.9 Hz, 1H), 4.48 (d, J=5.9 Hz, 1H), 4.35 (d,J=5.9 Hz, 1H), 4.15 (ddd, (J=10.1, 4.8, 4.0 Hz, 1H), 3.67 (m, 2H), 1.40(s, 3H), 1.27 (s, 3H), 1.04 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 135.74,135.67, 130.34, 130.29, 128.17, 128.15, 117.54, 102.81, 86.74, 86.52,82.30, 65.47, 26.47, 26.03, 24.30.

Step B5-O-[tert-Butyl(diphenyl)silyl)-2,3-O-(1-methylethylidene)-D-ribitol

To a cooled solution (0° C.) of5-O-[tert-butyl(diphenyl)silyl)-2,3-O-(1-methylethylidene)-D-glycero-pentofuranose(17 g, 39.7 mmol), preparation of which was described in the previousstep, in methanol (200 mL) was added NaBH₄ (1.65 g, 43.6 mmol). Thereaction mixture was kept stirring at 0° C. for 30 min before it warmedup to ambient temperature. Concentrated acetic acid was added dropwiseto adjust pH to 7.0. The solvent was removed in vacuo, and the residuewas dissolved in DCM (200 mL). The solution was washed with sat. NaHCO₃(300 mL) and water (3×500 mL). The organic layer was dried withanhydrous sodium sulfate, filtered and evaporated to dryness. Theresidue was purified by flash chromatography using 30% ethyl acetate inhexane to obtain 17 g (39.5 mmol, 100%) of the pure product. LCMS: forC₂₄H₃₄O₅Si calculated 430.2. found 453.1 [M+Na]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 7.71 (m, 4H), 7.44 (m, 6H), 4.22 (m, 2H), 3.76 (m, 6H), 3.58(ddd, J=11.8, 6.7, 5.3 Hz, 1H), 3.29 (dd, J=6.8, 5.3 Hz, 1H), 1.27 (s,6H), 1.03 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 135.75, 135.71, 130.06,130.05, 128.00, 127.96, 117.52, 77.86, 76.25, 70.09, 66.11, 60.73,27.41, 26.40, 24.86.

Step C1,4-Anhydro-5-O-[tert-Butyl(diphenyl)silyl)-2,3-O-(1-methylethylidene)-D-ribitol

A mixture of5-O-[tert-butyl(diphenyl)silyl)-2,3-O-(1-methylethylidene)-D-ribitol (17g, 39.5 mmol) and p-toluenesulfonyl chloride (9.03 g, 47.4 mmol) inpyridine (100 mL) was heated at 100° C. for 16 h. After cooling toambient temperature, the solvent was removed in vacuo. The residue wasdissolved in DCM (200 mL), washed successively with aqueous sat. NaHCO₃(300 mL) and water (3×500 mL). The organic layer was dried withanhydrous sodium sulfate, filtered and evaporated to dryness. Theresidue was purified by flash chromatography using 5-10% ethyl acetatein hexane to obtain 13 g (31.5 mmol, 80%) of the pure product. LCMS: forC₂₄H₃₄O₅Si calculated 412.2. found 413.2 [M+H]⁺. ¹H NMR (600 MHz, CD₃CN)δ: 7.67 (m, 4H), 7.44 (m, 6H), 4.80 (dd, J=5.4, 4.5 Hz, 1H), 4.74 (dd,J=6.2, 0.7 Hz, 1H), 4.00 (t, J=4.1 Hz, 1H) 3.93 (dd, J=10.3, 4.2 Hz,1H), 3.82 (d, J=10.2 Hz, 1H), 3.67 (t, J=4.3 Hz, 2H), 1.41 (s, 3H), 1.29(s, 3H), 1.02 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 135.71, 135.64,130.19, 130.14, 128.09, 117.53, 85.18, 82.79, 81.64, 73.83, 64.73,26.41, 26.14, 24.27.

Step D 1,4-Anhydro-2,3-O-(1-methylethylidene)-D-ribitol

To a mixture of1,4-anhydro-5-O-[tert-Butyl(diphenyl)silyl)-2,3-O-(1-methylethylidene)-D-ribitol(49 g, 119 mmol) and CsF (54.1 g, 356 mmol) in THF (200 mL) was addedTBAF (1 M in THF, 17.81 mL, 17.81 mmol). The mixture was stirred at 70°C. for 16 h, cooled to ambient temperature, diluted with methanol (50mL) and stirred for 10 min. The solvent was co-evaporated in vacuo withtoluene (3×50 mL) and the residue was dissolved in DCM (60 mL).Purification by flash chromatography using 80-90% ethyl acetate inhexane gave 18.5 g (106 mmol, 89%) of the pure product. LCMS: forC₈H₁₄O₄ calculated 174.1. found 175.0 [M+H]⁺. ¹H NMR (600 MHz, CD₃CN) δ:4.76 (ddd, J=5.9, 4.4, 1.4 Hz, 1H), 4.59 (dd, J=6.3, 1.1 Hz, 1H), 3.93(dt, J=6.2, 1.0 Hz, 1H), 3.85 (m, 2H), 3.46 (m, 2H), 2.82 (s, 1H), 1.41(s, 3H), 1.28 (s, 3H). ¹³C NMR (600 MHz, CD₃CN) δ: 117.54, 85.27, 82.48,81.38, 72.29, 61.32, 26.11, 24.21.

Step E 1,4-Anhydro-D-ribitol

To a cold (0° C.) solution of TFA (44.2 mL, 574 mmol) and water (11 mL)was added 1,4-anhydro-2,3-O-(1-methylethylidene)-D-ribitol (10 g, 57.4mmol) and stirred at ambient temperature for 2 h when TLC showed nostarting material. Toluene (30 mL) was added and the volatiles wereremoved under reduced pressure while temperature was kept below 30° C.Co-evaporation with toluene was repeated two more times. The residue waspurified by flash chromatography using 10-15% methanol in DCM to obtain6.15 g (45.9 mmol, 80%) of the pure product. LCMS: for C₅H₁₀O₄calculated 134.1. found 157.0 [M+Na]⁺. ¹H NMR (600 MHz, CD₃CN) δ: 4.07(dd, J=8.7, 5.0 Hz, 1H), 3.93 (dd, J=9.6, 4.9 Hz, 1H), 3.89 (t, J=5.8Hz, 1H), 3.60 (m, 3H), 3.47 (dd, J=12.6, 5.6 Hz, 1H), 3.41 (s, 1H). ¹³CNMR (600 MHz, CD₃CN) δ: 117.58, 83.17, 72.70, 72.03, 71.18, 62.27.

Step F 1,4-Anhydro-3,5-O-(di-tert-butylsilanylidene)-D-ribitol

To a solution of 1,4-anhydro-D-ribitol (6.11 g, 45.6 mmol) in pyridine(40.5 mL, 501 mmol) at 0° C. was added dropwisedi-tert-butylsilylbis(trifluoromethanesulfonate (22.07 g, 50.1 mmol)through an addition funnel with vigorous stifling over a period of 30min. The mixture was slowly warmed to ambient temperature andcontinuously stirred for 1 h. The solvent was removed in vacuo, and theresidue was partitioned between water (50 mL) and DCM (50 mL). Theorganic layer was dried with anhydrous sodium sulfate and concentrated.The residue was purified by flash chromatography using 10-30% ethylacetate in hexane to obtain 10.4 g (37.9 mmol, 83%) of the pure product.LCMS: for C₁₃H₂₆O₄Si calculated 274.2. found 275.1 [M+H]⁺. ¹H NMR (600MHz, CD₃CN) δ: 4.30 (dd, J=7.8, 3.7 Hz, 1H), 4.22 (ddd, J=4.1, 4.1, 1.7Hz, 1H), 4.10 (ddd, J=10.3, 4.3, 1.3 Hz, 1H), 3.82 (m, 3H), 3.69 (s,1H), 3.02 (t, J=1.7 Hz 1H), 1.04 (s, 9H), 1.01 (s, 9H). ¹³C NMR (600MHz, CD₃CN) δ: 117.54, 78.84, 74.48, 73.16, 69.81, 68.23, 27.05, 26.77,26.31.

Intermediate 2

Step A 3′,5′-O-(di-tert-butylsilanylidene)inosine

Under the protection of N₂, to a solution of inosine (40 g, 149 mmol) inanhydrous DMF (400 mL) at 0° C. was added dropwisedi-tert-butylsilyl-bistriflate (53.1 mL, 164 mmol). After the abovesolution was stirred for 30 min at 0° C., imidazole (50 g, 746 mmol) wasadded and the reaction mixture was stirred at room temperature for 1 h.The solvents were removed under reduced pressure and the solid residuewas triturated with dichloromethane and washed with H2O and collected.The 54 g (132 mmol, 89%) product was generated and applied in the nextstep without further purification. LCMS: for C₁₈H₂₈N₄O₅Si calculated408.1. found 409.1 [M+H]⁺. ¹H NMR (600 MHz, CD₃OD) δ: 8.14 (s, 1H), 8.02(s, 1H), 5.97 (s, 1H), 4.66 (dd, J=9.3, 4.9 Hz, 1H), 4.57 (d, J=4.8 Hz,1H), 4.41 (dd, J=8.9, 4.9 Hz, 1H), 4.15 (ddd, J=10.3, 10.3, 5.0 Hz, 1H),4.06 (dd, J=9.4, 9.2 Hz, 1H), 1.096 (s, 9H), 1.055 (s, 9H). ¹³C NMR (600MHz, CD₃OH) δ: 145.59, 139.77, 91.56, 76.41, 74.79, 73.99, 67.389,26.75, 26.483.

Step B 2′-O-acetyl-3′,5′-O-(di-tert-butylsilanylidene)inosine

Under the protection of N₂, Acetic anhydride was added drop-wise to amixture of 3′,5′-O-(di-tert-butylsilanylidene)inosine (5.3 g, 12.97mmol), DMAP (0.317 g, 2.59 mmol), and anhydrous pyridine (25 mL, 309mmol) in anhydrous DMF (50 mL) at 0° C. The mixture was stirred at 0° C.for 10 min subsequently under room temperature for 2 h. The reaction wasquenched with methanol (40 mL) and evaporated to dryness. The solidresidue was dissolved into EtOAc and washed three times with saturatedsodium bicarbonate solution. Organic layer was dried over MgSO4 thenevaporated to produce 5.6 g (12.87 mmol, 99%) pure compound. LCMS: forC₂₀H₃₀N₄O₆Si calculated 450.2. found 451.0 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 7.90 (s, 1H), 7.90 (s, 1H), 6.03 (s, 1H), 5.63 (d, J=5.4 Hz,1H), 4.81 (ddd, J=7.2, 5.4, 1.7 Hz, 1H), 4.40 (m, 1H), 4.06 (m, 2H),2.12 (s, 3H) 1.075 (s, 9H), 1.021 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ:145.69, 139.07, 117.55, 88.46, 75.20, 74.874, 67.15, 26.90, 26.40,26.63.

Step C9-[2-O-acetyl-3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-({[2,4,6-tri(propan-2-yl)phenyl]sulfonyl}oxy)-9H-purine

Under the protection of N₂, to a solution of2′-O-acetyl-3′,5′-O-(di-tert-butylsilanylidene)inosine (5.6 g, 12.87mmol) in 120 mL of anhydrous dichloromethane were added DMAP (0.315 g,2.57 mmol), triethylamine (7.18 mL, 51.5 mmol) and2,4,6-tri-isopropylbenzenesulfonyl chloride (11.7 g, 16 mmol,). After 2h of stirring at room temperature the mixture was diluted with 100 mL ofdichloromethane, washed three times with saturated sodium bicarbonatesolution and one time with brine. The organic layers were combined,dried over MgSO₄ and evaporated to dryness. Resulting residue waspurified by column chromatography (gradient: hexane with EtOAc 0-40%)and 3.97 g (5.54 mmol, 43%) pure compound obtained. LCMS: forC₃₅H₅₂N₄O₈SSi calculated 716.3. found 717.2 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 8.47 (s, 1H), 8.30 (s, 1H), 7.35 (s, 2H), 6.14 (s, 1H), 5.68(d, J=5.2 Hz, 1H), 4.90 (ddd, J=7.2, 5.4, 1.7 Hz, 1H), 4.39 (m, 1H),4.23 (m, 2H), 4.07 (m, 2H), 2.97 (m, 1H), 2.12 (s, 3H), 1.23 (m, 18H),1.07 (s, 9H), 1.01 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 169.56, 155.41,154.73, 154.01, 151.30, 151.18, 145.17, 131.43, 124.56, 123.70, 117.56,88.75, 75.33, 74.76, 74.69, 67.07, 34.25, 29.93, 26.92, 26.65, 23.83,22.89, 22.38, 20.18, 20.08.

Step D9-[2-O-acetyl-3,5-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Under the protection of N₂, to a solution of9-[2-O-acetyl-3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-({[2,4,6-tri(propan-2-yl)phenyl]sulfonyl}oxy)-9Hpurine (3.97 g, 5.54 mmol) in anhydrous dioxane (100 mL) were addedDABCO (1.24 g, 11.07 mmol) and 8 g of dry 3 Å molecular sieves. After 30min of stirring at room temperature 2-(trimethylsilyl)ethanol (3.97 mL,27.7 mmol) and DBU (2.08 mL, 13.84 mmol) were added and the reaction wasstirred at room temperature for 2 h. The mixture was then filtered offand the filtrate was evaporated to dryness. The residue was re-dissolvedinto 100 mL dichloromethane and washed with 100 mL saturated sodiumbicarbonate solution. After NaHCO₃ layer was extracted with 100 mLdichloromethane three times, organic layers were combined, dried overMgSO₄ and evaporated to dryness. Resulting residue was purified bycolumn chromatography (gradient: hexane with EtOAc 0-50%) and 2.8 g(5.08 mmol, 92%) pure compound obtained. LCMS: for C₂₅H₄₂N₄O₆Si₂calculated 550.3. found 551.3 [M+H]⁺. ¹H NMR (600 MHz, CD₃CN) δ: 8.44(s, 1H), 8.01 (s, 1H), 6.10 (s, 1H), 5.71 (d, J=5.2 Hz, 1H), 4.97 (dd,J=9.2, 5.3 Hz, 1H), 4.67 (m, 2H), 4.40 (dd, J=8.1, 4.0 Hz, 1H), 4.07 (m,2H), 2.12 (s, 3H), 1.22 (m, 2H), 1.09 (s, 9H), 1.01 (s, 9H), 0.08 (s,9H). ¹³C NMR (600 MHz, CD₃CN) δ: 169.677, 152.28, 141.98, 117.58, 88.54,75.18, 74.86, 74.77, 67.18, 65.44, 58.84, 26.92, 26.71, 26.65, 22.40,21.59, 20.20, 20.08, 17.21.

Step E9-[3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

To a 0° C. 40 mL methanol solution of9-[2-O-acetyl-3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(2.6 g, 4.72 mmol), 0.5M sodium methoxide (28.3 ml, 14.16 mmol) inmethanol was added drop-wise. After mixture was stirred at 0° C. for 10min, reaction was quenched with addition of ammonium chloride (1.01 g,18.88 mmol) and stirred at room temperature for 5 min. The mixture wasfiltered and the filtrate was evaporated. The residue was dissolved into50 mL EtOAc and the organic layer was washed three times with 50 mLsaturated sodium bicarbonate solution and one time with 50 mL H2O. Thenafter the organic layer was dried with MgSO4 and evaporated to producethe 1.70 g (3.34 mmol, 70.8%) product. LCMS: for C₂₃H₄₀N₄O₅Si₂calculated 508.3. found 509.3 [M+H]⁺. ¹H NMR (600 MHz, CD₃OD) δ: 8.46(s, 1H), 8.34 (s, 1H), 6.03 (s, 1H), 4.71 (m, 2H), 4.64 (d, J=4.8 Hz,1H), 4.57 (s, 1H), 4.40 (dd, J=9.0, 5.0 Hz, 1H), 4.16 (ddd, J=10.0,10.0, 5.1 Hz, 1H), 4.06 (dd, J=10.4, 9.2 Hz, 1H), 1.25 (m, 2H), 1.11 (s,9H), 1.06 (s, 9H), 0.09 (s, 9H). ¹³C NMR (600 MHz, CD₃OD) δ: 152.24,142.40, 91.68, 76.37, 74.79, 73.78, 67.41, 65.47, 26.77, 26.49, 17.24.

Intermediate 3

Step A9-[2-O-(4-bromobenzyl)-3,5-o-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Under the protection of N₂,9-[3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(10.00 g, 19.66 mmol) was dissolved into 100 mL anhydrous acetonitrilewith 3 Å molecular sieves (5 g). After the mixture was cooled to 0° C.,2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine(BEMP) (13.48 g, 49.1 mmol) followed immediately by 4-bromobenzylbromide (14.74 mL, 59.00 mmol) were added with exclusion of moisture.The reaction was then warmed up gradually from 0° C. to room temperatureand stirred at room temperature for 16 h. The mixture was quenched withmethanol (15 mL) and the solvents were evaporated under the reducedpressure. The residue was purified with flash chromatography using 20%etheyl acetate in hexane to obtain 12.8 g (18.89 mmol, 96%) of the pureproduct. LCMS: for C₃₀H₄₅BrN₄O₅Si₂ calculated 677.8. found 679.3 [M+H]⁺.¹H NMR (600 MHz, CD₃CN) δ: 8.47 (s, 1H), 8.09 (s, 1H), 7.51 (d, J=8.5Hz, 2H), 7.35 (d, J=8.3 Hz, 2H), 6.14 (s, 1H), 4.96 (d, J=12.3, 1H),4.77 (m, 2H), 4.69 (m, 2H), 4.47 (d, J=4.9 Hz, 1H), 4.43 (dd, J=9.1, 5.0Hz, 1H), 4.21 (ddd, J=10.3, 10.3, 5.0 Hz, 1H), 4.08 (dd, J=10.2, 9.4 Hz,1H), 1.23 (m, 2H), 1.12 (s, 9H), 1.04 (s, 9H), 0.10 (s, 9H). ¹³C NMR(600 MHz, CD₃CN) δ: 152.15, 141.39, 131.48, 129.56, 117.59, 89.67,80.75, 76.83, 74.94, 71.81, 67.39, 65.39, 26.98, 26.70, 17.21, −2.14.

Step B9-[2-O-(4-bromobenzyl)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Hydrogen fluoride-pyridine (7.43 g, 75.0 mmol) was carefully dilutedwith pyridine (30.3 mL, 375 mmol) and then added drop wise to a solutionof9-[2-O-(4-bromobenzyl)-3,5-o-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(7.43 g, 74.97 mmol) in THF (100 mL) at 0° C. The mixture was slowlywarmed up to room temperature and stirred for 2 h. The reaction wasquenched with methanol (10 mL) and then evaporated to dryness. Theresidue was purified by flash chromatography using 90% ethyl acetate inhexanes to obtain 8.8 g (16.37 mmol, 87%) of the pure product. LCMS: forC₂₂H₂₉BrN₄O₅Si calculated 537.5. found 539.1 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 8.30 (s, 1H), 8.09 (s, 1H), 7.15 (m, 2H), 6.93 (d, J=8.2 Hz,2H), 5.93 (d, J=7.3 Hz, 1H), 5.27 (dd, J=10.5, 2.9 Hz, 1H), 4.72 (m,2H), 4.64 (m, 2H), 4.45 (s, 1H), 4.34 (d, J=12.6 Hz, 1H), 4.18 (d, J=1.6Hz, 1H), 3.78 (dt, J=12.7, 2.5 Hz, 1H), 3.68 (ddd, J=12.6, 10.4, 2.2 Hz,1H), 3.56 (d, J=3.0 Hz, 1H), 1.28 (m, 2H), 0.12 (s, 9H). ¹³C NMR (600MHz, CD₃CN) δ: 151.36, 142.65, 136.94, 131.07, 129.97, 121.25, 117.68,117.50, 88.34, 87.92, 79.34, 71.24, 69.97, 65.70, 62.69, 17.26, −2.12.

Step C9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-o-(4-bromobenzyl)-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine

To a solution of9-[2-O-(4-bromobenzyl)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(8.75 g, 16.28 mmol) in pyridine (50 mL, 618 mmol) was added4,4′-dimethoxytrityl chloride (6.62 g, 19.54 mmol). The mixture wasstirred at ambient temperature for 2 h. Solvent was then evaporated invacuo, and the residue purified by flash chromatography using 35-40%ethyl acetate in hexane to obtain 11.26 g (13.41 mmol, 82%) of the pureproduct. LCMS: for C₄₃H₄₇BrN₄O₇Si calculated 839.8. found 814.4 [M+H]⁺.¹H NMR (600 MHz, CD₃CN) δ: 8.27 (s, 1H), 8.06 (s, 1H), 7.42, (m, 2H),7.27 (m, 7H), 7.10 (d, J=8.3 Hz, 2H), 6.82 (m, 4H), 6.08 (d, J=5.1 Hz,1H), 4.70 (m, 4H), 4.54 (d, J=12.5 Hz, 1H), 4.47 (dd, J=9.6, 5.1 Hz,1H), 4.17 (dd, J=8.5, 4.3 Hz, 1H), 3.77 (s, 6H), 3.57 (d, J=5.4 Hz, 1H),3.33 (d, J=4.3 Hz, 2H), 1.26 (m, 2H), 0.11 (s, 9H). ¹³C NMR (600 MHz,CD₃CN) δ: 160.86, 158.86, 158.85 151.84, 145.26, 141.75, 137.24, 136.05,136.01, 131.32, 130.26, 130.20, 129.92, 128.21, 127.05, 122.13, 121.35,117.56, 113.23, 87.19, 86.37, 84.46, 80.09, 71.52, 69.95, 65.31, 63.51,55.13, 17.29, −2.08

Intermediate 4

Intermediate 4 was synthesized using procedures analogous to thosedescribed for synthesis of Intermediate 3 except that in Step A, insteadof para-bromobenzyl bromide, ortho-bromobenzyl bromide was used.

Example 11,4-Anhydro-2-O-benzyl-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol

Step A1,4-Anhydro-2-O-benzyl-3,5-O-(di-tert-butylsilanylidene)-D-ribitol

To a solution of 1,4-Anhydro-3,5-O-(di-tert-butylsilanylidene)-D-ribitol(2.00 g, 7.29 mmol) in acetonitrile (20 mL) cooled at 0° C. was addedmolecular sieves (3 g), benzyl bromide (4.33 mL, 36.4 mmol) and2-tert-butylimino-2-diethylamino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine(4.00 g, 14.58 mmol). The reaction mixture was slowly warmed to ambienttemperature and stirred for 16 h. The reaction was quenched with theaddition of MeOH (5 mL) and stirred for 5 min. The solid was filteredoff with a layer of Celite. The filtrate was concentrated in vacuo, andthe residue purified by flash chromatography using 5-10% ethyl acetatein hexane to obtain 2.30 g of the pure product (6.31 mmol, 87%). LCMS:for C₂₀H₃₂O₄Si calculated 364.2. found 365.2 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 7.35 (m, 4H), 7.27 (t, J=7.1 Hz, 1H), 4.91 (d, J=12.0 Hz, 1H),4.63 (d, J=11.9 Hz, 1H), 4.31 (dd, J=8.9, 4.6 Hz, 1H), 4.12 (m 2H), 3.97(dd, J=9.5, 4.2 Hz, 1H), 3.87 (ddd, J=10.0, 10.0, 4.7 Hz, 1H), 3.80 (m,2H), 1.05 (s, 9H), 1.01 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 128.44,127.55, 117.57, 79.74, 77.24, 73.52, 73.06, 72.08, 68.27, 27.02, 26.76.

Step B 1,4-Anhydro-2-O-benzyl-D-ribitol

To a mixture of1,4-anhydro-2-O-benzyl-3,5-O-(di-tert-butylsilanylidene)-D-ribitol (2.60g, 7.13 mmol) and potassium fluoride (4.14 g, 71.3 mmol) in THF (30 mL)was added TBAF (1 M in THF, 3.63 mL, 3.63 mmol). The mixture was stirredat 50° C. for 16 h. It was then cooled to ambient temperature andfiltered though a layer of Celite, and washed with ACN (50 mL). Thefiltrate was concentrated in vacuo, and the residue purified by flashchromatography using 25-30% ethyl acetate in hexane to obtain 1.05 g(4.68 mmol, 66%) of the pure product. LCMS: for C₁₂H₁₆O₄ calculated224.1. found 225.0 [M+H]⁺ and 247.0 [M+Na]⁺. ¹H NMR (600 MHz, CD₃CN) δ:7.37 (m, 4H), 7.30 (tt, J=7.0, 1.6 Hz, 1H), 4.61 (dd, J=22.6, 11.9 Hz,2H), 4.00 (m, 2H), 3.92 (dd, J=9.5, 5.1 Hz, 1H), 3.75 (dd, J=9.6, 3.7Hz, 1H), 3.66 (ddd, J=4.5, 4.5, 3.6 Hz, 1H), 3.60 (dd, J=12.0, 3.0, 1H),3.48 (m, 1H), 3.06 (d, J=6.3 Hz 1H), 2.80 (s, 1H). ¹³C NMR (600 MHz,CD₃CN) δ: 128.56, 128.02, 127.86, 117.57, 83.98, 78.77, 71.84, 71.67,70.17, 62.21.

Step C1,4-Anhydro-2-O-benzyl-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]D-ribitol

To a solution of 1,4-anhydro-2-O-benzyl-D-ribitol (1.0 g, 4.46 mmol) inpyridine (20 mL, 247 mmol) was added 4,4′-dimethoxytrityl chloride (4.53g, 13.38 mmol). The mixture was stirred at ambient temperature for 2 h.Solvent was then evaporated in vacuo, and the residue purified by flashchromatography using 25-30% ethyl acetate in hexane to obtain 1.65 g(3.13 mmol, 70%) of the pure product. LCMS: for C₃₃H₃₄O₆ calculated526.2. found 549.2 [M+H]⁺. ¹H NMR (600 MHz, CD₃CN) δ: 7.42, (m, 2H),7.37 (m, 4H), 7.30 (m, 7H), 7.21 (tt, J=7.3, 1.1 Hz, 1H), 6.85 (m, 4H),4.61 (d, J=3.4 Hz, 2H), 4.05 (dd, J=9.9, 4.7 Hz, 1H), 4.01 (m, 2H), 3.81(m, 2H), 3.76 (s, 2H), 3.76 (s, 6H), 3.17 (dd, J=10.2, 3.2 Hz, 1H), 3.00(dd, J=10.2, 5.0, 1H), 2.97 (s, 1H). ¹³C NMR (600 MHz, CD₃CN) δ: 158.84,130.21, 128.59, 128.27, 128.01, 127.88, 126.98, 117.53, 113.22, 82.73,78.59, 72.28, 71.87, 70.21, 64.44, 55.11.

Step D1,4-Anhydro-2-O-benzyl-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-D-ribitol

To a cooled solution (0° C.) of1,4-anhydro-2-O-benzyl-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]D-ribitol(1.60 g, 3.04 mmol) and N,N-diisopropylethylamine (1.59 mL, 9.11 mmol)in DCM (20 mL), 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (1.44g, 6.08 mmol) and 1-methylimidazole (0.12 mL, 1.519 mmol) were added.The reaction mixture was warmed to ambient temperature and stirred for90 min. The reaction was then quenched with methanol (5 mL) and stirredfor 5 min. The solvent was then removed in vacuo, and the residue waspurified by flash chromatography using 20% ethyl acetate in hexane toobtain 0.94 g (1.293 mmol, 42.6%) of the pure product. LCMS: forC₄₂H₅₁N₂O₇P calculated 726.3. found 727.3 [M+H]⁺ and 749.4 [M+Na]⁺ ³¹PNMR (400 MHz, CD₃CN) δ: 149.94, 149.70.

Example 22′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]inosine

Step A9-[2-O-benzyl-3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Under the protection of N₂,9-[3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.65 g, 3.24 mmol) was dissolved into 50 mL anhydrous acetonitrile with3 Å molecular sieves (3 g). After the mixture was cooled to 0° C.,2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine(BEMP) (1.88 mL, 6.49 mmol) followed immediately by benzyl bromide (1.94mL, 16.22 mmol) were added with exclusion of moisture. The reaction wasthen warmed up gradually from 0° C. to room temperature and stirred atroom temperature for 2 h. The mixture was quenched with methanol (5 mL)and the solvents were evaporated under the reduced pressure. The residuewas purified by column chromatography (gradient: Hexane with EtOAc0-50%) to produce 1.72 g (2.87 mmol, 88%) final product. LCMS: forC₃₀H₄₆N₄O₅Si₂ calculated 598.3. found 599.1 [M++H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 8.45 (s, 1H), 8.01 (s, 1H), 7.40 (m, 2H), 7.33 (m, 2H), 7.27(m, 1H), 6.13 (s, 1H), 4.98 (d, J=12.0 Hz, 1H), 4.77 (m, 2H), 4.67 (m,2H), 4.46 (d, J=4.9 Hz, 1H), 4.41 (dd, J=9.1, 5.0 Hz, 1H), 4.20 (ddd,J=10.1, 10.1, 4.9 Hz, 1H), 4.06 (dd, J=10.3, 9.3 Hz, 1H), 1.22 (m, 2H),1.11 (s, 9H), 1.04 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 152.12, 141.40,128.50, 127.79, 127.69, 117.52, 117.15, 89.73, 80.76, 76.91, 74.96,72.67, 67.42, 65.33, 27.00, 26.71, 17.21, −0.85, −2.14.

Step B 2′-O-benzylinosine

To a mixture of9-[2-O-benzyl-3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.72 g, 2.87 mmol) and potassium fluoride (1.50 g, 25.80 mmol) in THF(30 mL) was added TBAF (1 M in THF, 2.87 mL, 2.87 mmol). The mixture wasstirred at 55° C. for 2 h. After it was cooled to room temperature, thereaction was filtered and washed with THF (30 mL). The filtrate wasevaporated under the reduced pressure and the residue was purified byflash chromatography (gradient: dichloromethane with methanol 0-8%) toproduce 1.00 g (2.79 mmol, 97%) product. LCMS: for C₁₇H₁₈N₄O₅ calculated358.1. found 358.9 [M+H]⁺. (600 MHz, CD₃OD) δ: 8.17 (s, 1H), 7.92 (s,1H), 7.10 (m, 5H), 6.03 (d, J=6.3 Hz, 1H), 4.72 (d, J=12.4 Hz, 1H), 4.50(dd, J=6.1, 5.1 Hz, 1H), 4.46 (d, J=12.4 Hz, 1H), 4.42 (dd, J=4.8, 2.9Hz, 1H), 4.15 (dd, J=5.8, 2.9 Hz, 1H), 3.81 (dd, J=12.4, 2.8 Hz, 1H),3.71 (dd, J=12.4, 2.9 Hz, 1H). ¹³C NMR (600 MHz, CD₃OD) δ: 148.00,145.36, 139.90, 137.53, 128.00, 127.88, 127.68, 87.82, 87.12, 80.38,72.15, 69.57, 61.93.

Step C 2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine

Under the protection of N₂, to a solution of 2′-O-benzylinosine (1.0 g,2.79 mmol) in 10 mL pyridine was added 4,4′-dimethoxytrityl chloride(2.36 g, 6.98 mmol). The mixture was stirred at room temperature for 2h. The reaction was diluted with 100 mL dichloromethane and quenchedwith 100 mL saturated NaHCO3 solution. After the aqueous layer wasextracted with dichloromethane (100 mL) three times, the organic layerswere combined and evaporated. The residue was purified by flashchromatography (gradient: hexane with EtOAc 0-45%, with 1% triethylaminein both hexane and EtOAc) to produce 1.80 g (2.72 mmol, 98%) product.LCMS: for C₃₈H₃₆N₄O₇ calculated 660.3. found 661.2 [M+H]⁺. ¹H NMR (600MHz, CD₃CN) δ: 7.83 (s, 1H), 7.80 (s, 1H), 7.39 (m, 2H), 7.23 (m, 12H),6.82 (m, 4H), 6.02 (d, J=5.0 Hz, 1H), 4.71 (d, J=12.2 Hz, 1H), 4.59 (d,J=12.2 Hz, 1H), 4.54 (dd, J=5.1, 5.1 Hz, 1H), 4.42 (dd, J=9.6, 4.8 Hz,1H), 4.14 (dd, J=8.4, 4.2 Hz, 1H), 3.64 (m, 1H), 3.57 (m, 1H). ¹³C NMR(600 MHz, CD₃CN) δ: 158.88, 156.87, 145.48, 145.18, 138.51, 136.09,135.98, 130.23, 128.50, 128.24, 128.16, 128.08, 128.07, 128.04, 128.00,127.08, 117.58, 117.08, 117.04, 113.264, 86.91, 84.49, 80.70, 72.29,69.84, 63.55, 62.88, 55.15, 52.76.

Step D2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]inosine

Under the protection of N₂, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.21 mL, 5.45 mmol) was addeddrop-wise to a 0° C. anhydrous tetrahydrofuran (12 mL) solution of2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (1.80 g,2.72 mmol), N,N-diisopropylethylamine (1.42 mL, 8.17 mmol)), and1-methylimidazole (0.11 mL, 1.36 mmol). The reaction was graduallywarmed to room temperature and stirred for 60 min. The reaction was thenquenched with methanol (5 mL) and stirred for 5 min. After the solventwas removed under the reduced pressure, the residue was purified byflash chromatography (gradient: methylene chloride with methanol 0-4%,with 1% triethylamine in both methylene chloride and methanol) toproduce 1.83 g (78%, 2.13 mmol) final product. LCMS: for C47H53N6O8Pcalculated 860.4. found 861.3 [M+H]⁺ and 884.4 [M+Na]⁺. ³¹P NMR (600MHz, CD₃CN) δ: 150.82, 150.55.

Example 3

Step A9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[4-(pyridin-4-yl)benzyl]-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Palladium (II) acetate (58.8 mg, 0.262 mmol) and butyldi-1-adamantylphosphine (169 mg, 0.472 mmol) were added to1,2-dichloroethane (10 mL) at ambient temperature and stirred for 20min. The solvent was then removed in vacuo. To this residue was added asolution of9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-o-(4-bromobenzyl)-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine(Intermediate 3, 2.20 g, 2.62 mmol) and pyridine-4-boronic acid (580 mg,4.72 mmol) in 2-methyl-THF (40 mL), followed by the addition ofsaturated aqueous solution of potassium carbonate (2.17 g, 15.7 mmol).The mixture was heated at 80° C. and stirred for 16 h. After cooled toambient temperature, the mixture was partitioned between water (50 mL)and DCM (50 mL). The aqueous layer was washed with DCM (20 mL) threetimes. The combined organic layers were dried with anhydrous sodiumsulfate and then solvent was evaporated in vaduo. The residue waspurified with flash chromatography using 85% ethyl acetate in hexane toobtain 1.90 g (2.27 mmol, 87%) of the pure product. LCMS: forC₄₈H₅₁N₅O₇Si calculated 838.0. found 838.6 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 8.61 (m, 2H), 8.21 (s, 1H), 8.04 (s, 1H), 7.54, (m, 2H), 7.46(m, 2H), 7.40 (m, 2H), 7.23 (m, 7H), 6.80 (m, 4H), 6.08 (d, J=5.5 Hz,1H), 4.79 (m, 2H), 4.54 (m, 4H), 4.20 (dd, J=8.3, 4.3 Hz, 1H), 3.74 (s,6H), 3.33 (d, J=4.4 Hz, 2H), 1.16 (m, 2H), 0.09 (s, 9H). ¹³C NMR (600MHz, CD₃CN) δ: 160.73, 158.85, 158.83, 151.79, 151.72, 150.37, 147.45,145.27, 141.90, 139.01, 137.30, 136.08, 136.03, 130.25, 130.19, 128.75,128.20, 128.02, 127.03, 126.80, 122.09, 121.59, 117.88, 117.58, 117.40,113.22, 87.23, 86.35, 84.62, 79.82, 71.88, 70.00, 65.16, 63.59, 55.11,17.19, −2.13.

Step B 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-2-o-[4-(pyridin-4-yl)benzyl]-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine

To a cooled solution (0° C.) of9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-o-[4-(pyridin-4-yl)benzyl]-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.85 g, 2.21 mmol) and N,N-diisopropylethylamine (856 mg, 6.62 mmol) inDCM (20 mL), 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (1.05 g,4.42 mmol) and 1-methylimidazole (91 mg 1.10 mmol) were added. Thereaction mixture was warmed to ambient temperature and stirred for 90min. The reaction was then quenched with methanol (5 mL) and stirred for5 min. The solvent was then removed in vacuo, and the residue waspurified by flash chromatography using 65% ethyl acetate in hexane toobtain 1.91 g (1.84 mmol, 83%) of the pure product. LCMS: forC₅₇H₆₈N₇O₈PSi calculated 1038.3. found 1039.4 [M+H]⁺ ³¹P NMR (600 MHz,CD₃CN) δ: 150.70, 150.65.

Example 4

Step A9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-o-[4-(1,3-oxazol-2-yl)benzyl]-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Tris[dibenzylideneacetone]dipalladium(0) (131 mg, 0.143 mmol) and[4-(N,N-dimethylamino)phenyl]di-tert-butylphosphine (152 mg, 0.572 mmol)were added to THF (10 mL) at ambient temperature and stirred for 30 min.To this mixture was then added a solution of9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-o-(4-bromobenzyl)-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine(Intermediate 3, 800 mg, 0.953 mmol) and 2-(tri-N-butylstannyl)oxazole(1.02 g, 2.86 mmol) in THF (15 mL). The mixture was heated at 50° C. andstirred for 16 h. After cooled to ambient temperature, the mixture wasfiltered through a layer of Celite. The filtrate was concentrated invacuo. The residue was purified with flash chromatography using 55-60%ethyl acetate in hexane to obtain 735 mg (0.888 mmol, 88%) of the pureproduct. LCMS: for C₄₆H₄₉N₅O₈Si calculated 828.0. found 828.3 [M+H]⁺. ¹HNMR (600 MHz, CD₃CN) δ: 8.22 (s, 1H), 8.05 (s, 1H), 7.88 (s, 1H), 7.77(d, J=8.2 Hz, 2H), 7.41 (d, J=8.0 Hz, 2H), 7.25 (m, 7H), 6.80 (m, 4H),6.09, (d, J=5.5 Hz, 1H), 4.78 (m, 2H), 4.58 (m, 4H), 4.19 (dd, J=8.4,4.3 Hz, 1H), 3.76 (s, 6H), 3.59 (d, J=5.2 Hz, 2H), 3.33 (d, J=4.5 Hz,2H), 1.21 (m, 2H), 0.10 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 158.82,151.74, 145.21, 141.78, 140.29, 139.61, 136.05, 136.00, 130.21, 130.17,128.58, 128.40, 128.18, 127.98, 127.00, 126.00, 117.54, 117.07, 113.19,87.14, 86.34, 84.55, 80.02, 71.85, 69.98, 65.20, 63.53, 55.08, 17.18,−2.13.

Step B

To a cooled solution (0° C.) of9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-o-[4-(1,3-oxazol-2-yl)benzyl]-β-D-ribofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.63 g, 1.97 mmol) and N,N-diisopropylethylamine (763 mg, 5.91 mmol) inDCM (20 mL), 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (932 mg,3.94 mmol) and 1-methylimidazole (81 mg 0.984 mmol) were added. Thereaction mixture was warmed to ambient temperature and stirred for 90min. The reaction was then quenched with methanol (5 mL) and stirred for5 min. The solvent was then removed in vacuo, and the residue waspurified by flash chromatography using 55% ethyl acetate in hexane toobtain 1.57 g (1.53 mmol, 78%) of the pure product. LCMS: forC₅₅H₆₆N₇O₉PSi calculated 1027.4. found 1028.5 [M+H]⁺ ³¹P NMR (600 MHz,CD₃CN) δ: 150.69, 150.64.

Example 5

Step A9-[3,5-O-(di-tert-butylsilanylidene)-β-D-erythro-pentofurnaosyl-2-ulose]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

To a solution of9-[3,5-O-(di-tert-butylsilanylidene)-β-D-ribofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine.(50 g, 98 mmol), and DCC (58.8 g, 285 mmol) in DMSO (200 ml) and Benzene(250 ml), Dichloroacetic acid (2.8 ml, 33 mmol) was added drop-wise at0° C. under the protection of N₂. The reaction was warmed gradually toRT and was stirred for 4 hours. The reaction mixture was partitionedbetween H₂O (800 ml) and EtOAc (800 ml) and separated. After the organiclayer was washed H₂O (800 ml) three times and dried with MgSO4. Thesolvent was evaporated to produce the 51 g crude product. The crudeproduct was a mixture of the desired 2′-ketone and its corresponding2′-geminal diol. It was applied into the next step of synthesis withoutany further purification. LCMS9-[3,5-O-(di-tert-butylsilanylidene)-β-D-erythro-pentofurnaosyl-2-ulose]-6-[2-(trimethylsilyl)ethoxy]-9H-purine:for C₂₃H₃₈N₄O₅Si₂ calculated 506.7. found 539.2 [M+Na]⁺. LCMS(4aR,6R,7aR)-2,2-di-tert-butyl-6-{6-[2-(trimethylsilyl)ethoxy]-9H-purin-9-yl}dihydro-4H-furo[3,2-d][1,3,2]dioxasiline-7,7(6H)-diol:for C₂₃H₄₀N₄O₆Si₂ calculated 524.7. found 525.1 [M+H]⁺.

Step B9-[3,5-O-(di-tert-butylsilanylidene)-β-D-arabinofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

To a solution of crude9-[3,5-O-(di-tert-butylsilanylidene)-β-D-erythro-pentofurnaosyl-2-ulose]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(51 g, 97 mmol) in Ethanol (150 ml), Sodium borohydride (7.4 g, 194mmol) was added under the protection of N₂. The reaction was stirredunder RT for 2 hours. After the reaction was quenched with aqueous HCl,the solvent was removed under vacuum and the residue was partitionedbetween water (500 ml) and DCM (500 ml) and separated. The aqueous layerwas washed DCM (500 ml) three times, all of the organic layers werecombined and dried with MgSO4 and evaporated. Resulting residue waspurified by column chromatography (gradient: hexane with EtOAc 0-50%)and 25.9 g (50.9 mmol, 51.9% two steps) pure compound obtained. LCMS:for C₂₃H₄₀N₄O₅Si₂ calculated 508.8. found 509.1 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 8.42 (s, 1H), 8.23 (s, 1H), 6.40 (d, J=7.1, 2H), 4.68 (m, 2H),4.61 (t, J=8.9 Hz, 1H), 4.51 (dd, J=8.2, 7.3 Hz, 1H), 4.33 (dd, J=9.1,5.3 Hz, 1H), 4.18 (m, 1H), 3.85 (m, 1H), 1.22 (m, 2H), 1.09 (s, 9H),1.01 (s, 9H), 0.07 (s, 9H). ¹³C NMR (600 MHz, CD₃CN) δ: 160.77, 152.10,152.01, 143.19, 120.77, 83.67, 80.25, 75.08, 73.69, 67.16, 65.38, 26.80,26.43, 22.34, 19.75, 17.24.

Step C9-[2-O-benzyl-3,5-O-(di-tert-butylsilanylidene)-β-D-arabinofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Under the protection of N₂,9-[3,5-O-(di-tert-butylsilanylidene)-β-D-arabinofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.95 g, 3.83 mmol) was dissolved into 50 mL anhydrous acetonitrile with3 Å molecular sieves (3 g). After the mixture was cooled to 0° C.,2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine(BEMP) (2.22 mL, 7.67 mmol) followed immediately by benzyl bromide (2.30mL, 19.16 mmol) were added with exclusion of moisture. The reaction wasthen warmed up gradually from 0° C. to room temperature and stirred atroom temperature for 2 h. The mixture was quenched with methanol (5 mL)and the solvents were evaporated under the reduced pressure. The residuewas purified by column chromatography (gradient: Hexane with EtOAc0-50%) to produce 2.20 g (3.67 mmol, 96%) final product. LCMS: forC₃₀H₄₆N₄O₅Si₂ calculated 598.88. found 599.27 [M+H]⁺. ¹H NMR (600 MHz,CD₃CN) δ: 8.42 (s, 1H), 8.12 (s, 1H), 7.16 (m, 3H), 6.92-6.91 (m, 2H),6.52 (d, J=7.0 Hz, 1H), 4.72 (t, J=8.9 Hz, 1H), 4.68-4.65 (m, 2H), 4.54(d, J=12.0 Hz, 2H), 4.48 (dd, J=8, 7.3 Hz, 2H), 4.35 (d, J=9.1, 5.3 Hz,1H), 4.3 (d, J=12.1 Hz, 1H), 4.15-4.12 (m, 1H), 3.88 (ddd, J=10, 10, 5.2Hz, 1H), 1.22 (m, 2H), 1.10 (s, 9H), 1.03 (s, 9H). 0.08 (s, 9H). ¹³C NMR(600 MHz, CD₃CN) δ: 152.43, 152.13, 142.86, 137.56, 128.38, 127.83,127.50, 106.38, 81.73, 81.63, 79.95, 73.26, 72.37, 67.17, 65.31, 27.10,26.68, 17.24, −2.12.

Step D9-(2-O-benzyl-β-D-arabinofuranosyl)-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Hydrogen fluoride-pyridine (1.18 mL, 9.17 mmol) was carefully dilutedwith pyridine (5 mL) and then added drop wise to a solution of9-[2-O-benzyl-3,5-O-(di-tert-butylsilanylidene)-β-D-arabinofuranosyl]-6-[2-(trimethylsilyl)ethoxy]-9H-purine(2.2 g, 3.67 mmol) in THF (100 mL) at 0° C. The mixture was stirred at0° C. for 15 min. The reaction was quenched with methanol (10 mL) andthen evaporated to dryness. The residue was diluted with 50 mLdichloromethane and was washed with water (100 mL) three times, theorganic layers was evaporated to obtain 1.60 g (3.49 mmol, 95%) of thepure product. LCMS: for C₂₂H₃₀N₄O₅Si calculated 458.583. found 459.1[M+H]⁺. ¹H NMR (600 MHz, CD₃OD) δ: 8.46 (s, 1H), 8.40 (s, 1H), 7.10-7.06(m, 3H), 6.82 (d, J=6.6 Hz, 2H), 6.53 (d, J=5.5 Hz, 1H), 4.73-4.70 (m,2H), 4.57-4.54 (m, 2H), 4.47 (t, J=5.6, 1H), 4.27 (m, 2H), 3.93 (m, 1H),3.86-3.79 (m, 2H), 1.26 (dd, J=9.6, 7.2 Hz, 2H), 0.10 (s, 9H). ¹³C NMR(600 MHz, CD₃OD) δ: 152.02, 142.97, 127.98, 127.50, 127.26, 83.92,83.72, 83.51, 73.30, 72.61, 65.44, 60.70, 17.28, −2.59.

Step E9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-benzyl-β-D-arabinofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Under the protection of N₂, to a solution of9-(2-O-benzyl-β-D-arabinofuranosyl)-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.60 g, 3.49 mmol) in 20 mL pyridine was added 4,4′-dimethoxytritylchloride (2.13 g, 6.28 mmol). The mixture was stirred at roomtemperature for 2 h. The reaction was diluted with 100 mLdichloromethane and quenched with 100 mL saturated NaHCO3 solution.After the aqueous layer was extracted with dichloromethane (100 mL)three times, the organic layers were combined and evaporated. Theresidue was purified by flash chromatography (gradient: hexane withEtOAc 0-100%, with 1% triethylamine in both hexane and EtOAc) to produce1.18 g (1.55 mmol, 44.4%) product. LCMS: for C₄₃H₄₈N₄O₇Si calculated760.9. found 762.5 [M+H]⁺. ¹H NMR (600 MHz, CD₃CN) δ: 8.39 (s, 1H), 8.10(s, 1H), 7.42-7.40 (m, 2H), 7.29-7.13 (m, 10H), 6.86-6.85 (m, 2H),6.81-6.77 (m, 4H), 6.50 (d, J=5.5 Hz, 1H), 4.69-4.65 (m, 2H), 4.49-4.45(m, 2H), 4.24-4.21 (m, 2H), 4.02 (m, 1H), 3.8 (dd, J=10.4, 6.3 Hz, 1H),3.29 (dd, J=10.4, 3.5 Hz, 1H), 1.22 (m, 2H). 0.09 (s, 1H). ¹³C NMR (600MHz, CD₃CN) δ: 160.82, 152.01, 142.44, 130.24, 130.21, 128.39, 128.25,128.01, 127.86, 127.61, 127.05, 113.21, 83.56, 82.87, 82.37, 74.31,72.54, 65.26, 63.56, 55.10, 17.27, −2.13.

Step F 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-2-O-benzyl-β-D-arabinofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine

Under the protection of N₂, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.69 mL, 3.10 mmol) was addeddrop-wise to a 0° C. anhydrous tetrahydrofuran (12 mL) solution of9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-benzyl-β-D-arabinofuranosyl}-6-[2-(trimethylsilyl)ethoxy]-9H-purine(1.18 g g, 1.55 mmol), N,N-diisopropylethylamine (0.81 mL, 4.65 mmol)),and 1-methylimidazole (0.06 mL, 0.78 mmol). The reaction was graduallywarmed to room temperature and stirred for 60 min. The reaction was thenquenched with methanol (5 mL) and stirred for 5 min. After the solventwas removed under the reduced pressure, the residue was purified byflash chromatography (gradient: hexane with EtOAc 0-50%, with 1%triethylamine in both hexane and EtOAc) to produce 1.41 g (94.6%, 1.47mmol) final product. LCMS: for C₅₂H₆₅N₆O₈PSi calculated 961.167. found961.6 [M+H]⁺. ³¹P NMR (600 MHz, CD₃CN) δ: 150.65, 150.24.

Example 6N-acetyl-2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]adenosine

The title compound was synthesized using a procedure analogous to thatdescribed under Example 2, with appropriate protecting groups present inthe heterocyclic ring. LCMS: for C₄₆H₅₃N₄O₉P calculated 836.36. found836.20. ³¹P NMR (600 MHz, CDCl₃) δ: 150.76, 150.55.

Example 72′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]uridine

The title compound was synthesized using a procedure analogous to thatdescribed under Example 2, with appropriate protecting groups present inthe heterocyclic ring. LCMS: for C₅₄H₅₈N₇O₈P calculated 963.41. found965.8 [M+H]⁺ ³¹P NMR (600 MHz, CDCl₃) δ: 151.27, 151.76.

Example 82′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-N-(2-methylpropanoyl)-guanosine

The title compound was synthesized using a procedure analogous to thatdescribed under Example 2, with appropriate protecting groups present inthe heterocyclic ring. LCMS: for C₅₄H₅₈N₇O₈P calculated 945.42. found945.40. ³¹P NMR (600 MHz, CDCl₃) δ: 150.76, 150.53.

Example 9N-Acetyl-2′-O-benzyl-5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3′-O-[(2-cyanoethoxy)(dipropan-2-ylamino)phosphanyl]-N-(2-methylpropanoyl)-cytidine

The title compound was synthesized using a procedure analogous to thatdescribed under Example 2, with appropriate protecting groups present inthe heterocyclic ring. LCMS: for C₄₈H₅₆N₅O₉P calculated 877.38. found879.0 [M+H]⁺ ³¹P NMR (600 MHz, CDCl₃) δ: 150.66, 150.42.

What is claimed is:
 1. A method for evaluating 1 or more chemicalmodifications in an siRNA, said method comprising: a) providing a siRNAoligomer with 1 or more sugar un-modified universal base containingnucleosides; b) measuring position-specific data to create a baselinedata; c) providing a siRNA oligomer with 1 or more sugar modifieduniversal base containing nucleosides at the same position of theun-modified universal base(s) of step a); d) measuring position-specificdata, wherein a change in the position-specific data from step d)relative to the baseline data is indicative of siRNA function.
 2. Themethod of claim 1, wherein the position-specific data isposition-specific knockdown data.
 3. The method of claim 1, wherein theevaluation of the position-specific data comprises detecting target mRNAdegradation.
 4. The method of claim 3, wherein the target mRNAdegradation is detected by quantitative PCR.
 5. The method of claim 1,wherein the universal base is a hydrogen atom, inosine, modifiedadenine, modified guanine, modified uracil, modified cytosine, thymine,modified thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine,naphthyl, 3-nitropyrrole, imidazole-4-carboxamide, 5-nitroindole,nebularine, pyridone, or pyridinone.
 6. The method of claim 1, whereinthe universal base is inosine.
 7. The method of claim 1, wherein theuniversal base is anhydroribitol.
 8. The method of claim 1, wherein thesugar-modified universal base comprises a 2′-O-ribose modification. 9.The method of claim 8, wherein the 2′-O-ribose modification is a2′-O-benzyl, 2′-O-methylene-(β-naphthyl),2′-O-(2-difluoromethoxybenzyl), or 2′-O-methylene-(4-pyridyl)modification.
 10. The method of claim 1, wherein the siRNA is a doublestranded siRNA.
 11. The method of claim 10, wherein the siRNA comprisesoverhangs.
 12. The method of claim 1, wherein measuringposition-specific data in step b) comprises contacting the siRNAoligomer of step a) with a target mRNA.
 13. The method of claim 12,which further comprises detecting target mRNA degradation.
 14. Themethod of claim 1, wherein measuring position-specific data in step d)comprises contacting the siRNA oligomer of step c) with a target mRNA.15. The method of claim 14, which further comprises detecting targetmRNA degradation.
 16. The method of claim 1, wherein measuringposition-specific data in step b) comprises contacting a cell with thesiRNA oligomer of step a).
 17. The method of claim 1, wherein measuringposition-specific data in step d) comprises contacting a cell with thesiRNA oligomer of step c).
 18. The method of claim 1, which comprisescomparing the position specific data from step d) to the baseline data.